CICLO XXI COORDINATORE Prof. Pier Andrea BOREA Fischetti PhD... · 1 List of abbreviations 3...

Università degli Studi di Ferrara

DOTTORATO DI RICERCA IN FARMACOLOGIA E ONCOLOGIA MOLECOLARE

Joint Ph.D. programme between the Universities of Leicester (UK) and Ferrara (Italy)

CICLO XXI

COORDINATORE Prof. Pier Andrea BOREA

NOCICEPTIN/ORPHANIN FQ RECEPTOR LIGANDS: PHARMACOLOGICAL STUDIES

Settore Scientifico Disciplinare BIO/14 Dottorando Tutori Dr. Carmela Fischetti Dr. Girolamo Calo’

Prof. David G. Lambert

Anni 2006/2008

1

List of abbreviations 3

Abstract 6

Summary 7

1. INTRODUCTION

1.1 Orphan G-protein coupled receptors and the reverse 10 pharmacology approach

1.2 The NOP receptor 13 1.3 Identification of nociceptin/orphanin FQ 16 1.4 Metabolism of N/OFQ 20 1.5 NOP receptor and N/OFQ localization 21 1.6 Cellular effects of N/OFQ 23 1.7 Biological effects of N/OFQ 26 1.8 Pharmacology of NOP receptors 44 1.9 Aims 59

2. MATERIALS & METHODS

2.1 Drugs and reagents 60 2.2 Buffer composition 61

2.3 In vitro studies

CHO expressing the recombinant NOP/DOP/MOP/KOP receptors 2.3.1 Cell harvesting and membrane preparation 62 2.3.2 Displacement binding assay 63 2.3.3 [35S]GTPγS stimulation binding assay 63 2.3.4 Protein assay 64

Calcium mobilization assay 2.3.5 Experimental protocols 64 2.3.6 Cell counting 65 2.3.7 Instruments 66

Isolated tissues 2.3.8 Tissue preparation 68 2.3.9 Experimental protocols 68 2.3.10 Instruments 68

2.4 In vivo studies

Experimental protocol 2.4.1 Animals 70 2.4.2 Tail withdrawal assay 70

2.5 Data analysis and terminology 71

2

3. RESULTS & DISCUSSION

3.1 Pharmacological profile of NOP receptors coupled to calcium 74 signalling via the chimeric protein Gαqi5

3.2 Further studies on the pharmacological features of the 84 nociceptin/orphanin FQ receptor ligand ZP120

3.3 Pharmacological characterization of the nociceptin/orphanin FQ 94 receptor non-peptide antagonist Compound 24

3.4 Blending of chemical moieties of NOP receptor ligands: 105 identification of a novel antagonist

3.5 General conclusions 113

Bibliography 119

3

List of abbreviations

aa amino acid Ac acetyl Ach acetylcholine Aib amino isobutyric acid ANOVA One-Way Analyses of Variance APN Aminopeptidase N Arg Arginine AUC area under curve bNST bovine Nocistatin BSA bovine serum albumin cAMP cyclic adenosine monophosphate CPP conditioned place preference CHO chinese hamster ovary CNS central nervous system Compound 24 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4’-piperidin-1-yl]propyl} Compound 35 (D)-1-Benzyl-pyrrolidine-2-carboxylic acid {3-[4-(2,6-dichloro-

phenyl)-piperidin-1-yl]-propyl}-amide CRF corticotropin release factor DA dopamine DAG diacylglycerol DOP delta opioid peptide DPDPE [D-Pen2,D-Pen5]enkephalin DPN diprenorphine EDTA ethylenediamine-tetraacetic acid EL extracellular loop EP 24.11 endopeptidase 24.11 EP 24.15 endopeptidase 24.15 ERK extracellular signal-regulated kinase FCS fetal calf serum GABA γ-aminobutyric acid GDP guanosin diphosphate GPCR G-protein coupled receptor gpI guinea pig ileum GTP guanosin 5’-triphosphate GTPγS guanosine-5’-O-(3-thiotriphosphate) [35S]GTPγS guanosine-5’-[γ-35S]thiophosphate 5-HT serotonin HBSS Hanks’ Balanced Salt Solution HEPES 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid HTS high throughput screening i.c.v. intracerebroventricular(ly) i.p. intraperitoneo(ly) IP3 inositol 1,4,5-trisphosphate i.t. intrathecal(ly) i.v. intravenous(ly) IUPHAR International Union of Pharmacology (acronym) III-BTD (3S,6S,9R)-2-oxo-3-amino-7-thia-1-aza-bicyclo[4.3.0]nonane-9-

carboxylic acid

4

(±)J-113397 (±)trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one

JTC-801 N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride

KOP kappa opioid peptide MCOPPB 1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-

benzimidazole MOP mu opioid peptide mRNA messenger ribonucleic acid mVD mouse vas deferens NA noradrenaline NalBzOH naloxone benzoyhlydrazone NNC 63-0532 (8-Naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-

spiro[4.5]dec-3-yl)-acetic acid methyl ester N/OFQ Nociceptin/orphanin FQ NOP receptor Nociceptin/orphanin FQ peptide receptor NOP(+/+), NOP(-/-) NOP receptor knockout and wildtype NSB non specific binding NST nocistatin oGPCR orphan G-protein coupled receptor Oprl 1 gene NOP receptor gene ORL-1 receptor Opioid receptor-like 1 PAG periaqueductal gray PCPB 2-(3,5-dimethylpiperazin-1-yl)-1-[1-(1-methylcyclooctyl)piperidin-

4-yl]-1H-benzimidazole PCR polymerase chain reaction PLC phospholipase C ppN/OFQ prepronociceptin ppN/OFQ(-/-) prepronociceptin gene deficient PVN paraventricular nucleus Ro-65-6570 (8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one) Ro-64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-

1,3,8-triaza-spiro[4,5]decan-4-one] SAR structure-activity relationship RT-PCR reverse transcriptase polymerase chain reaction rVD rat vas deferens SB-612111 (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-

6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol SCH 221510 8-[bis(2-Methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-

3-ol s.e.m. standard error of the mean TM transmembrane TTX tetrodotoxin TRK-820 ((-)-17-cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6b-[N-

methyl-trans-3-(3-furyl)acrylamide]morphinan hydrochloride) UFP-101 [Nphe1Arg14Lys15]N/OFQ-NH2 UFP-102 [(pF)Phe4Arg14Lys15]N/OFQ-NH2 UFP-103 [Phe1ψ(CH2-NH)Gly2(pF)Phe4Arg14Lys15]N/OFQ-NH2 UFP-111 [Nphe1(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 UFP-112 [(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2

5

UFP-113 [Phe1ψ(CH2-NH)Gly2(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 ZP120 Ac-RYYRWKKKKKKK-NH2

6

Abstract (University of Leicester Format)

Nociceptin/orphanin FQ receptor ligands: pharmacological studies

The neuropeptide nociceptin/orphanin FQ (N/OFQ) selectively binds and activates

the N/OFQ peptide (NOP) receptor. At the cellular level N/OFQ inhibits cAMP

accumulation and Ca2+ conductance and stimulates K+ currents. N/OFQ regulates several

biological functions both at central (pain, locomotion, memory, emotional responses, food

intake) and peripheral (airways, cardiovascular, genitourinary and gastrointestinal systems)

sites. Potent and selective NOP ligands are now required for investigating the roles played

by NOP receptors in pathophysiological studies and for firmly defining the therapeutic

indications of NOP receptor ligands.

A novel assay to screen NOP receptor ligands has been validated with a large panel

of ligands: the Gαqi5 chimeric protein has been used to force the NOP receptor to signal

through the Ca2+ pathway in CHO cells. [Ca2+]i levels were monitored using the

fluorometer FlexStation II. Data are in general agreement with classical Gi driven assay

systems.

The NOP peptide partial agonist, ZP120 was extensively characterized in vitro

using electrically stimulated isolated tissues (mouse and rat vas deferens) and in vivo with

the tail withdrawal assay. The selective involvement of the NOP receptor in the actions of

ZP120 has been demonstrated in NOP(-/-) mice studies.

A detailed pharmacological characterization of the recently identified non-peptide

antagonist Compound 24 has been performed. Moreover in the context of a SAR study on

Compound 24, a novel NOP ligand named Compound 35 was identified. Compound 24

and Compound 35 bound the human recombinant NOP receptor expressed in CHOhNOP cell

membranes with high affinity (pKi values 9.62 and 9.14, respectively). Our findings

derived from functional studies on CHOhNOP and bioassay studies on native receptors

demonstrated that Compound 24 and Compound 35 behave as potent, competitive and

selective non-peptide NOP antagonists. Finally, the NOP antagonist properties of

Compound 24 have been confirmed in vivo in the mouse tail withdrawal assay.

7

Summary

N/OFQ and its receptor share high sequence similarity with opioid peptides,

particularly with dynorphin A, and their receptors. However N/OFQ and dynorphin use

distinct molecular pathways to bind and activate their cognate receptors. Thus, N/OFQ and

NOP receptor represent a novel peptidergic system, which is pharmacologically distinct

from the opioid systems. At cellular level N/OFQ inhibits cAMP accumulation and Ca2+

conductance and stimulates K+ currents, and in vivo it modulates a variety of biological

functions: nociception, food intake, memory processes, anxiety, locomotor activity,

gastrointestinal motility, cardiovascular and renal functions, micturition and cough

reflexes.

The pharmacological characterization of new ligands for this peptide/receptor

system has been the main aim of the studies performed during this Ph.D. program. In close

collaboration with Prof Lambert’s group (University of Leicester) and with Medicinal

Chemistry group of Prof. Salvadori (University of Ferrara), we performed a series of

studies on novel ligands for the NOP receptor. The most important areas are summarized

as below:

1) Pharmacological profile of NOP receptors coupled with calcium signalling via the

chimeric protein Gαqi5

In this study the Gαqi5 protein was used to force the human NOP receptor to signal

through the Ca2+ pathway in CHO cells. [Ca2+]i levels were monitored using the

FlexStation II fluorometer and the Ca2+ dye Fluo 4 AM. Concentration response curves

were generated with a panel of full and partial agonists while NOP antagonists were

assessed in inhibition response curves.

The following rank order of potency of antagonists was measured: SB-612111 > J-

113397 = Trap-101 > UFP-101 > [Nphe1]N/OFQ(1-13)-NH2 >> naloxone, which is in

good agreement with the literature. The rank order of potency of full and partial agonists is

also similar to that obtained in previous studies with the exception of a panel of ligands

(UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Gαqi5 -

NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow

kinetics of interaction with the NOP receptor, as demonstrated by bioassay experiments.

This study demonstrated that the FlexStation II - Gαqi5 - NOP receptor calcium assay

8

represents an adequate and useful screening tool for NOP receptor ligands, particularly for

antagonists.

2) Further studies on the pharmacological features of the nociceptin/orphanin FQ

receptor ligand ZP120

In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in

mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further

investigate the pharmacological profile of ZP120 using mouse and rat preparations, J-

113397 and UFP-101, as well as NOP(-/-) mice. Electrically stimulated mouse and rat vas

deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies

the tail withdrawal assay was performed in wild type (NOP(+/+)) and NOP(-/-) mice. In

the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher

potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ

and ZP120 effects with similar pKB values (≈ 7.8). UFP-101 antagonized the actions of

N/OFQ (pKB values ≈ 7.3) but did not modify the effects of ZP120. The inhibitory effects

of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP(-/-)

mice. In NOP(+/+) mice subjected to the tail withdrawal assay, ZP120 (1 nmol) mimicked

the pronociceptive action of N/OFQ (10 nmol), producing longer lasting effects. The

effects of both peptides were absent in NOP(-/-) animals. The NOP receptor ligand ZP120

is a high potency NOP selective partial agonist able to evoke long-lasting effects; its

diverse antagonist sensitivity in comparison with N/OFQ may derive from different

modality of binding to the NOP receptor.

3) Pharmacological characterization of the nociceptin/orphanin FQ receptor non-

peptide antagonist Compound 24

Compound 24, 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4’-piperidin-1-yl]propyl}

pyrrolidine-2-carboxamide was recently identified as a NOP ligand. In this study, the in

vitro and in vivo pharmacological profile of Compound 24 was investigated. In vitro

studies were performed measuring receptor and [35S]GTPγS binding and calcium

mobilization in cells expressing the recombinant NOP receptor as well as using N/OFQ

sensitive tissues. In vivo studies were conducted using the tail withdrawal assay in mice.

Compound 24 produced a concentration-dependent displacement of [3H]N/OFQ binding to

9

CHOhNOP cell membranes showing high affinity (pKi 9.62) and selectivity (1000 fold) over

classical opioid receptors. Compound 24 antagonized with high potency the following in

vitro effects of N/OFQ: stimulation of [35S]GTPγS binding in CHOhNOP cell membranes

(pA2 9.98), calcium mobilization in CHOhNOP cells expressing the Gαqi5 chimeric protein

(pKB 8.73), inhibition of electrically evoked twitches in the mouse (pA2 8.44) and rat (pKB

8.28) vas deferens, and in the guinea pig ileum (pKB 9.12). In electrically stimulated

tissues, Compound 24 up to 1 µM did not modify the effects of classical opioid receptor

agonists. Finally in vivo, in the mouse tail withdrawal assay, Compound 24 at 10 mg/kg

antagonized the pronociceptive and antinociceptive effects of 1 nmole N/OFQ given

supraspinally and spinally, respectively. The present study demonstrated that Compound

24 is a pure, competitive, and highly potent non-peptide NOP receptor selective antagonist.

4) Blending of chemical moieties of NOP receptor ligands: identification of a novel

antagonist

In the present investigation, we performed a structure-activity analysis of

Compound 24, focussing on its N-benzyl D-Pro, amide bond, and benzoisofurane moieties.

This latter structure was substituted with moieties taken from known non-peptide NOP

receptor ligands. Twelve Compound 24 derivatives were synthesised and tested in binding

experiments performed on CHOhNOP cell membranes. Compound 24 displayed a pKi value

of 9.62 while the analogues modified on the N-benzyl D-Pro and amide bond moieties

showed very low affinities. In contrast, Compound 35 in which the benzoisofurane was

substituted with the 2,6-dichlorophenyl moiety of the NOP antagonist SB-612111, showed

high affinity (pKi 9.14). This novel compound was pharmacologically characterized in

various assays where it consistently behaved as a pure, potent (pA2 in the range 8.0 – 9.9),

competitive and NOP selective antagonist. Collectively the present results indicate that the

N-benzyl D-Pro and amide bond of Compound 24 are crucial for biological activity.

Moreover, a novel interesting NOP receptor antagonist was identified by blending

chemical moieties taken from different NOP receptor ligands.

10

1. INTRODUCTION

1.1 Orphan G-protein coupled receptors and the reverse pharmacology approach

G-protein coupled receptors (GPCRs) are one of the largest family of proteins that

are the main modulators of intercellular interactions and regulate activities in the human

body and in particularly in the central nervous system (CNS). There are numerous GPCRs

in living organisms, but the function of many is still unknown. The human genome

encompasses ~ 800 GPCRs, of which more than half are olfactory and/or taste GPCRs.

They are targets of most of the primary messengers including the neurotransmitters, all the

neuropeptides, the glycoprotein hormones, lipid mediators and other small molecules; thus

have considerable pharmaceutical interest. Drugs that are acting on GPCRs are used to

treat numerous disorders. More than 30 % of the approximately 500 clinically used drugs,

are modulators of GPCRs function, representing around 9 % of global pharmaceutical

sales, making GPCRs the most successful of any target class in terms of drug discovery

(Drews, 2000).

367 transmitter GPCRs have been identified within the human genome, the

majority of these GPCRs have been identified on the basis of their sequence similarities,

either by homology cloning or by bioinformatics analyses. Many of these receptors are

currently ‘orphans’.

The first step in the characterization of new orphan GPCRs is the search of the

activating ligand. As the genomes of most studied model organism have now been

sequenced, the process of discovery of GPCRs-ligand pairs has been reversed. Until

recently, neuropeptides have been traditionally identified either on the basis of their

chemical characteristics (Tatemoto et al., 1980) or of their effects in particular assay

systems (Erspamer et al., 1978). Although highly successful, these approaches had reached

a stand still by the mid 80’s.

Through DNA recombination techniques, it is now possible to transfect the

sequence of an orphan receptor of which the function is not yet known, into an appropriate

cellular expression system. This leads to the use of orphan receptors as baits to isolate their

natural ligands from mixtures of synthetic ligands, including known GPCR ligands,

naturally occurring bioactive molecules of unknown function and randomized compounds

in high-throughput screening (HTS). This approach has been named “reverse

pharmacology” (Figure 1). Thus, drug identification precedes the mechanistic

understanding of mode of action of the drug candidate. The expression system provides the

11

necessary trafficking and G-protein-signalling machinery to enable the successful

identification of the activating ligand. By exposing the transfected cell to a tissue extract

containing the natural ligand of the orphan receptor, a change in intracellular second

messengers will be induced and will serve as a parameter to monitor orphan receptor

ligand purification. Despite the logic of the theory, the process is not simple, since the

physical nature of the ligand and the type of the second messenger response that it will

generate, are unknown. However, structural features in an orphan GPCR will determine its

relationship to known receptors and will help in evaluating the nature of the receptor’s

ligand and its activity. Indeed, an orphan receptor which is related, even to a low degree, to

a particular receptor family has a higher probability of sharing a ligand of the same

physical nature and a coupling to similar G proteins. Notably this strategy has already led

to several significant discoveries. The orphan receptor strategy was first proven to be

successful with the discovery of the neuropeptide N/OFQ, the subject of this thesis, as the

endogenous ligand of the oGPCR ORL-1 (Meunier et al., 1995; Reinscheid et al., 1995).

Third party material removed

Figure 1. The orphan receptor strategy (Civelli et al.,TRENDS in Neurosciences Vol.24 No.4 April 2001). The orphan receptor strategy was developed to identify the natural ligands of orphan G-protein-coupled receptors (GPCRs) with the aim of discovering novel transmitters (defined in the main text). This strategy involves: (1) expression of the cloned orphan GPCR in an heterologous cell line; (2) exposure of this transfected cell line to a tissue extract that is expected to contain the natural ligand; (3) recording of the change in second messenger response elicited by activation of the orphan GPCR; (4) fractionation of the tissue extract and isolation of a surrogate, the active component; (5) determination of the chemical structure of the active component and (6) chemical synthesis of the active component and demonstration that it exhibits identical activity to that of the purified ligand.

12

This first successful example of orphan receptor strategy was followed by the

identification of other novel bioactive peptides such as: hypocretins and orexins, prolactin-

releasing peptide, apelin, ghrelin, melanin-concentrating hormone, urotensin II,

neuromedin U, metastin, neuropeptide B, neuropeptide W and neuropeptide S. Each of

these discoveries was a landmark in its field (Civelli, 2005). The success in GPCRs

deorphanization led to the approach being used by the pharmaceutical industry (Wise et

al., 2004), which had mastered the HTS of thousands of ligands. This led to thousands of

potential transmitters and unexpected ligands (also of non-peptide nature) being tested on

dozens of oGPCRs and a revival of the reverse pharmacology approach. Table 1

summarizes the transmitters of peptidic and non-peptidic nature identified as ligands of

oGPCRs after 1995 (Civelli, 2005).

Table 1. Transmitters identified as ligands of oGPCRs after N/OFQ; taken from Civelli (2005).


13

1.2 The NOP receptor

Pharmacological studies have defined at least three subtypes of opioid receptors,

termed μ, δ and κ receptors, that are involved in the mediation of the numerous effects,

like analgesia, respiratory depression, miosis, constipation, sensation of well being,

tolerance and dependence.

The nomenclature for the opioid receptors remains controversial. A 1996 review

and proposal for a novel nomenclature (Dhawan et al., 1996) based on guidelines from

NC-IUPHAR has not been widely accepted by the research community. The 1996 proposal

recommended replacement of the terms μ, δ, and κ with the terms OP3, OP1, and OP2,

respectively. However, in the three years or more since the publication of this

recommendation, almost all papers referring to opioid receptors have continued to use the

well-established Greek symbol nomenclature. Since Greek nomenclature gave many

problems in manuscript preparation and particularly WEB searches, this was substituted

with terminology more consistent with the overall guidelines of NC-IUPHAR that named

the opioid receptors as: DOP, MOP, and KOP (Cox et al., 2000).

Molecular cloning of the DOP receptor (Evans et al., 1992; Kieffer et al., 1992)

was soon followed by the cloning of the KOP and MOP receptors (Chen et al., 1993;

Yasuda et al., 1993). Further attempts to clone additional opioid receptor types and/or

subtypes, by hybridization screening at low stringency with opioid receptor cDNA probes,

or using probes generated by selective amplification of genomic DNA with degenerate

primers, led several laboratories to isolate a cDNA encoding a homologous protein with a

high degree of sequence similarity to the opioid receptors (Bunzow et al., 1994; Chen et

al., 1994; Fukuda et al., 1994; Lachowicz et al., 1995; Mollereau et al., 1994; Wang et al.,

1994).

The novel clone Opioid Receptor Like-1 (ORL-1) receptor displayed approximately

50 % identity with the traditional opioid receptors overall, with the transmembrane regions

showing even higher homologies of up to 80 %. Despite the close homology to the other

opioid receptors, opioid ligands displayed very low affinities towards ORL-1 receptor, thus

it was considered an orphan receptor. ORL-1 receptor is a typical GPCR with seven

predicted transmembrane domains (Figure 2).

14


Figure 2. Schematic representation of ORL-1 receptor from Topham et al. (1998). TM helices are numbered 1 to 7. E/IL: Extracellular/Intracellular Loop. Visible at the C-terminal of TM 6 is the Gln 286 (human receptor numbering) side chain.

ORL-1 was subsequently named NOP (Nociceptin/Orphanin FQ Peptide) receptor

according to the IUPHAR nomenclature (Cox et al., 2000). The ORL-1 receptor was

identified in different species and showed substantial sequence identities (>90%) between

species variants, namely the human (hNOP, (Mollereau et al., 1994)), rat (XOR1 (Wang et

al., 1994); ROR-C (Fukuda et al., 1994); LC132 (Bunzow et al., 1994), C3 (Lachowicz et

al., 1995)), mouse (MOR-C (Nishi et al., 1994)) and pig (NOP receptor, (Osinski et al.,

1999b)).

The human NOP receptor protein consists of 370 amino acids (Mollereau et al.,

1994) and contains seven transmembrane (TM) domains. The N-terminal 44 amino acids

contain 3 consensus sequences for N-linked glycosylation (Asn-X-Ser/Thr). There are also

sites for potencial phosphorylation by protein kinase A (in the third intracellular loop) and

protein kinase C (in the second intracellular loop and the C-terminal).

15


Figure 3. Alignment of the amino acid sequence of the rat NOP receptor (Hyp 8-1) with the amino acid sequences of the rat brain DOP, MOP and KOP. Sequences identical in at least 3 of 4 aligned sequences are boxed. Gaps in the alignment are indicated by a dash (-). Putative transmenbrane regions are underlined. Taken from Wick et al. (1994).

The NOP sequence has 57-58% aa (amino acid) identity to each of the rat MOP

(Chen et al., 1994), DOP (Fukuda et al., 1993) and KOP (Minami et al., 1993). This

percent identity is slightly lower than those obtained when the sequences of opioid

receptors are compared to each other (62-67%).

16

The conservation among the four receptors is highest (>70%) in the II, III, and VII

transmembrane domains, and approximately 50% in the I, V and VI, but significantly

lower (24%) in the IV. This high level of sequence conservation within the transmembrane

domains lends weight to the view that the NOP receptor contains a TM binding pocket that

is the structural equivalent of alkaloid binding pocket of the opioid receptors. Indeed, the

NOP receptor has retained the ability, with low affinity, to bind and/or respond to opioid

receptor ligands, agonist and/or antagonist such as etorphine and diprenorphine (Mollereau

et al., 1994), buprenorphine (Wnendt et al., 1999), lofentanil (Butour et al., 1997), and

naloxone benzoylhydrazone (Noda et al., 1998).

The NOP receptor gene, Oprl1, is located at the q13.2-13.3 region of the human

chromosome 20 (Peluso et al., 1998) and has been mapped to the distal region of the

mouse chromosome 2 (Nishi et al., 1994). In terms of intron-exon organization, the NOP

receptor gene is nearly identical to that of the MOP, DOP, and KOP receptors, suggesting

that the four genes have evolved from a common ancestor and hence belong to the same

family (Meunier, 1997). Indeed, the NOP receptor appears to be evolutionary as old as the

opioid receptors, since NOP receptor-like genomic sequences have been reported in teleost

(Darlison et al., 1997), in cartilaginous fish (Li et al., 1996), in sturgeon (Danielson et al.,

2001) and in zebra fish (Gonzalez-Nunez et al., 2003).

Although pharmacological studies have not firmly established the existence of NOP

receptor subtypes (Calo et al., 2000b), NOP receptor heterogeneity is still an open

question. NOP receptor heterogeneity may result from differential expression of NOP

splice variants. So far, five splice variants of NOP mRNA have been isolated. One,

identified in rat (Wang et al., 1994), encodes a NOP variant with an insertion (intron 5) in

the second extracellular loop. The second splice variant, exhibiting an in frame deletion of

15 nucleotides at the 3’ end of the TMD 1 coding region (Halford et al., 1995; Wick et al.,

1994), does encode a functional receptor and has already been isolated from human tissue

(Peluso et al., 1998). Further, insertions of exons 3 and 4 (Curro et al., 2001) after the first

coding exon (exon 2) in rats result in three additional splice variants (Pan et al., 1998),

which again encode truncated and not functional receptors.

1.3 Identification of nociceptin/orphanin FQ

In 1995 Meunier et al. and Reinsheid et al. simultaneously described N/OFQ as the

endogenous ligand for ORL-1, now known as NOP. CHO cells expressing the orphan

ORL-1 were used to identify the endogenous ligand. Based on structural similarities with

17

the known opioid receptors, both the chemical nature of the endogenous ligand (peptide)

and the consequences of receptor activation (inhibition of cyclic AMP) were assumed to be

similar to those of classical opioids. Consequently, cells were stimulated with forskolin to

activate adenylyl cyclase and increase intracellular cAMP. As a Gi/o-coupled orphan

receptor, endogenous agonists at this receptor will inhibit the formation of cAMP. Extracts

from rat (Meunier et al., 1995) or pig (Reinscheid et al., 1995) brain were screened.

Fractions that were able to inhibit the adenylyl cyclase activity were further fractionated

through reverse-phase high-performance liquid chromatography. The purification and mass

spectrometry analyses identified a heptadecapeptide (Figure 4), the sequence of which was

determined. The synthetic peptide was shown to have high affinity (in the nanomolar

range) and to strongly inhibit forskolin-induced accumulation of cAMP in CHO cells

expressing the NOP receptor (EC50 about 1 nM), while showing no activity in non

transfected cells (Meunier et al., 1995). Moreover, when tested in vivo by

intracerebroventricular (i.c.v.) injection in mice, the peptide induced hyperalgesia in the

hot plate (Meunier et al., 1995) and tail flick (Reinscheid et al., 1995) tests. Decrease in

the locomotor activity but no analgesia was also observed in the hot plate test (Reinscheid

et al., 1995).

The group of Meunier termed the novel peptide nociceptin, based on apparent pro-

nociceptive properties, while that of Reinscheid named it orphanin FQ, as ligand of an

orphan receptor, whose first and last amino acids are Phe (F) and Gln (Q), respectively.

N/OFQ shares sequence homologies with the opioid peptide ligand dynorphin A

(Figure 4); despite the structural similarities these peptides are functionally quite distinct.

N/OFQ has no significant affinity for any of the opioid receptors (Reinscheid et al., 1998).


Figure 4. Structural similarities between dynorphin A and N/OFQ amino acid sequences (Guerrini et al., 2000b).

18

The N-terminal tetrapeptide sequences (message domains) of the two peptides are

very similar, with the only difference of the first amino acid residue (Phe in N/OFQ and

Tyr in dynorphin A); the C-terminal parts (address domains) of the two molecules are both

enriched in positively charged residues, such as arginine and lysine, even if distributed in

different positions.

N/OFQ is a heptadecapeptide cleaved from the polypeptide precursor

preproN/OFQ (ppN/OFQ). ppN/OFQ consists of a 181 amino acids in the rat, 176 amino

acids in humans and 187 amino acids in the mouse (Mollereau et al., 1996; Nothacker et

al., 1996) (Figure 5). The ppN/OFQ gene, that has been isolated from human, mouse and

rat, is highly conserved in the three species.


Figure 5. Amino acid sequence for N/OFQ precursor, ppN/OFQ (Calo et al., 2000b).

Analysis of the nucleotide sequence of the preproN/OFQ gene revealed structural

and organisational characteristics very similar to those of the opioid peptide precursors, in

particular preproenkephalin and preprodynorphin, suggesting that these peptides derive

from a common ancestor (Mollereau et al., 1996; Nothacker et al., 1996).

The ppN/OFQ gene is located on human chromosome 8 (8p21) (Mollereau et al.,

1996). In the ppN/OFQ sequence there are several pairs of basic amino acids that present

possible sites of cleavage for precursor maturation or for transcriptional regulation (Zaveri

et al., 2000). Therefore, several biologically relevant peptides may derive from the N/OFQ

precursor (Figure 5). Apparently two additional peptides are excised from the same

precursor: orphanin FQ2 and nocistatin (Okuda-Ashitaka et al., 1998). None of them bind

19

to NOP receptor (Mollereau et al., 1996; Nothacker et al., 1996), and until now specific

receptors for them have not been identified. The peptide following the N/OFQ sequence, is

a heptadecapeptide terminating with the couple FQ (orphanin FQ2): it has been found to be

biologically active, stimulating locomotor activity in mice (Florin et al., 1997) inducing

antinociception both spinally and supraspinally (Rossi et al., 1998) and inhibiting

gastrointestinal transit (Rossi et al., 1998).

The second peptide, named nocistatin (NST), has been reported to act as a

functional antagonist of N/OFQ (Okuda-Ashitaka et al., 1998). In most studies, NST was

found to be inactive per se, but was able to reverse several effects of N/OFQ, such as

induction of allodynia after spinal administration in mice (Minami et al., 1998; Okuda-

Ashitaka et al., 1998), inhibition of glutamate release from rat brain slices (Nicol et al.,

1998), impairment of learning and memory in mice (Hiramatsu et al., 1999a), stimulation

of food intake in rats (Olszewski et al., 2000). Moreover, NST can, per se, cause

antinociception after i.c.v. administration in the rat carrageenan test (Nakagawa et al.,

1999) or after intratechal (i.t.) administration in the rat formalin test (Yamamoto et al.,

2001). Interestingly, nocistatin or its C-terminal hexapeptide exerts anxiogenic-like effects

in mice; in fact it has been reported that the C-terminal hexapeptide (the most conserved

region among species), administered i.c.v., exerts clear anxiogenic-like effects in mice, in

contrast to N/OFQ, that in the same experimental model, acts as an anxiolytic (Gavioli et

al., 2002). Very recent findings demonstrated that the opposite effects of N/OFQ and NST

on supraspinal pain modulation result from their opposing effects on the excitability of

central amygdala nucleus-periaqueductal gray projection (CeA-PAG) neurons.

Electrophysiological studies showed that N/OFQ hyperpolarized CeA-PAG projection

neurons by enhancing an inwardly rectifying potassium conductance. In contrast, NST

depolarized CeA-PAG neurons by causing the opening of TRPC cation channels via a

G(αq/11)-PLC-PKC pathway (Chen et al., 2009).

In vitro studies demonstrated that bovine nocistatin (bNST) inhibited the K+-

induced [3H]5-HT release from mouse cortical synaptosomes, displaying similar efficacy

but lower potency than N/OFQ; this inhibitory effect was not prevented either by the NOP

receptor antagonist UFP-101, or by the non-selective opioid receptor antagonist, naloxone.

In contrast to N/OFQ, bNST reduced [3H]5-HT release from synaptosomes obtained from

NOP receptor knockout mice (Fantin et al., 2007).

20

1.4 Metabolism of N/OFQ

Degradation of the N/OFQ peptide is principally via aminopeptidase N (APN),

which releases the inactive N/OFQ(2-17) and endopeptidase (EP), which cleaves a variety

of bonds to release inactive fragments, Figure 6 (Calo et al., 2000b). Endopeptidase 24.15

(EP 24.15) (Montiel et al., 1997) acts on the peptide bonds Ala7-Arg8, Ala11-Arg12, Arg12-

Lys13 and releases inactive compounds; endopeptidase 24.11 (EP 24.11) acts on the

cleavage site Lys13-Leu14 and plays a major role in the initial stage of N/OFQ metabolism

in mouse spinal cord (Sakurada et al., 2002). C-terminal degradation also leads to a

reduction in binding affinity of N/OFQ for NOP, loss of the 4 amino acids from the C-

terminal tail as in N/OFQ(1-13) results in a 30-fold reduction in potency (Butour et al.,

1997). However, amidation of C-terminus of N/OFQ(1-13) restores ligand affinity and

potency, consequently N/OFQ(1-13)-NH2 is the shortest sequence retaining the full

biological activity of the endogenous ligand (Guerrini et al., 1997).


Figure 6. N/OFQ metabolism by aminopeptidase N (APN) and endopeptidases (EP), from Calo et al. (2000b).

21

1.5 NOP receptor and N/OFQ localization

The regional distribution of N/OFQ and the NOP receptor have been well described

(Bunzow et al., 1994; Fukuda et al., 1994; Houtani et al., 2000; Lachowicz et al., 1995;

Letchworth et al., 2000; Mollereau et al., 1994; Neal et al., 1999a; Neal et al., 1999b;

Nothacker et al., 1996; O'Donnell et al., 2001; Riedl et al., 1996; Wick et al., 1995). These

series of publications provide detailed descriptions of the distribution of the NOP receptor,

mRNA and binding in the brain which are beyond the scope of this work. They report a

good correlation between receptor binding distributions and those seen with in situ

hybridization. Regions with NOP receptor binding typically express NOP mRNA as well,

although the levels of mRNA and NOP binding do not always closely match (Neal et al.,

1999a).

The NOP receptor is widely expressed in the CNS, in particular in the forebrain

(cortical areas, olfactory regions, limbic structures: hippocampus and amygdala, thalamus),

throughout the brainstem (central periaqueductal gray, substantia nigra, several sensory

and motor nuclei), and in both dorsal and ventral horns of the spinal cord (Mollereau et al.,

2000; Neal et al., 1999a). The distribution patterns have suggested the involvement of the

NOP receptor system in motor and balance control, reinforcement and reward, nociception,

stress response, sexual behaviour, aggression and autonomic control of physiological

processes (Neal et al., 1999a).

It is worthy of mention that NOP receptors co-express with MOP receptors in the

dorsal horn of the spinal cord, the hippocampal formation and the caudate putamen (Judd

et al., 1996; Letchworth et al., 2000) in the midbrain periaqueductal gray and the nucleus

raphe magnus (Houtani et al., 2000). Distribution of NOP does not always overlap that of

opioid receptors: these anatomical differences may provide a possible explanation for the

different in vivo actions of N/OFQ and opioids (Ikeda et al., 1998; Monteillet-Agius et al.,

1998; Sim et al., 1997).

Due to the diffuse distribution of N/OFQ peptide and NOP receptor, this novel

system is associated with a large number of physiological responses and probably

contributes to homeostasis by modulating neuronal circuitry (Mollereau et al., 2000). This

might explain why the NOP(-/-) mice do not display an obvious phenotype (other than the

unrestrained nociceptive response and dysregulation of hearing ability) (Nishi et al., 1997)

and also why pharmacological effects of N/OFQ are sometimes contradictory (e.g. in pain

and locomotor tests) depending on the locus of injection and dose (Mollereau et al., 2000).

22

The NOP receptor mRNA has also been identified in the peripheral nervous system

and several other organs. It is expressed in peripheral ganglia and in the immune system. It

has been detected in rat intestine, vas deferens, skeletal muscles and spleen (Wang et al.,

1994) in porcine gastrointestinal tract and kidney (Osinski et al., 1999b), in several guinea

pig ganglia (Fischer et al., 1998), also in rat retina and heart (Mollereau et al., 2000).

Peluso and colleagues (Peluso et al., 1998) were the first to describe the

distribution of NOP receptor transcripts in man, in different brain regions by RT-PCR

technique: the highest amplification was observed in cortical areas (the frontal and

temporal cortex), in the hypothalamus, mamillary bodies, the substantia nigra, and

thalamic nuclei. Transcripts have also been detected in limbic structures (the hippocampus

and amygdala), brainstem (the ventral tegmental area, the locus coeruleus) and the pituitary

gland. This distribution, which is similar to that of rodents, suggests the participation of the

NOP receptor in numerous human physiological functions, such as emotive and cognitive

processes, neuroendocrine and sensory regulation.

Berthele and colleagues (Berthele et al., 2003) studied the differential expression of

NOP receptors in the human brain (cortex, basal ganglia, hippocampal area and

cerebellum) by utilizing on-section ligand binding corroborated with mRNA detection on

parallel sections of the same brain tissues. In general, [3H]-N/OFQ ligand binding and

NOP receptor mRNA expression were widespread and indicative of a considerable high

NOP receptor expression in these anatomical regions. [3H]-N/OFQ ligand binding and

NOP mRNA expression studies showed that the highest amounts of NOP receptor were

observed in the cerebellum, in the cortex (cingulate and prefrontal cortex), in the striatum

(caudate nucleus and the putamen) and in the lamina II, followed by laminae III, V and VI,

in the principal neurons of the dentate gyrus and in the hippocampal area (Berthele et al.,

2003).

The localization of N/OFQ-immunoreactive fibers and terminals and/or the

localization of the ppN/OFQ mRNA correspond reasonably well with the NOP receptor.

Limbic areas highly express N/OFQ, in particular the bed nucleus of the stria terminals,

and the amygdala nuclei (Boom et al., 1999; Neal et al., 1999b). A matching pattern of

N/OFQ and NOP receptor expression in the human and rodent central nervous system has

been observed (Berthele et al., 2003; Peluso et al., 1998; Witta et al., 2004). As with the

receptor, N/OFQ immunoreactivity and mRNA levels detected using in situ hybridization

are closely correlated. N/OFQ is found in lateral septum, hypothalamus, ventral forebrain,

23

claustrum, mammillary bodies, amygdala, hippocampus, thalamus, medial habenula,

ventral tegmentum, substantia nigra, central gray, interpeduncular nucleus, locus coeruleus,

raphe complex, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and

reticular formation, as well as the ventral and dorsal horns of the spinal cord (Neal et al.,

1999b). Recently N/OFQ was immunolocalized in rat lateral and medial olivocochlear

efferents (Kho et al., 2006). Although N/OFQ and opioid peptides show a similar

distribution, they are not colocalized in nociceptive centres such as the dorsal horn, the

sensory trigeminal complex or the periaqueductal gray (Schulz et al., 1996).

In the periphery, mRNA of N/OFQ was detected in rat ovary, in human spleen,

lymphocytes, and fetal, but not adult kidney (Mollereau et al., 1996; Nothacker et al.,

1996). Furthermore, it has been shown that, under physiological conditions, N/OFQ is

present in the human plasma (~ 10 pg/ml) (Brooks et al., 1998). In several pathological

conditions such as postpartum depression (Gu et al., 2003), Wilson’s disease (Hantos et

al., 2002), hepatocellular carcinoma (Horvath et al., 2004) and in acute and chronic pain

states (Ko et al., 2002b), plasma levels of N/OFQ are increased. In contrast, lower N/OFQ

plasma levels have been observed in patients suffering from fibromyalgia syndrome

(Anderberg et al., 1998), cluster headache (Ertsey et al., 2004) and migraine without aura

(Ertsey et al., 2005). Recent findings indicate that plasma levels of N/OFQ are increased in

sepsis (Williams et al., 2008).

1.6 Cellular effects of N/OFQ

The cellular actions of the classical opioid receptors (MOP/KOP/DOP) and the

NOP receptor have been shown to be pertussis toxin sensitive and therefore couple to

inhibitory G-proteins i.e. G-proteins with Gi/o alpha subunits (Reinscheid et al., 1996). G-

proteins are membrane bound/associated heterotrimeric proteins composed of α, β, γ

subunits. There are four major classes of G proteins including Gi/Go, Gs, Gq.

Activation of NOP, similar to MOP, KOP, and DOP opioid receptors activation,

leads to: (i) closing of voltage sensitive calcium channels, (ii) stimulation of potassium

efflux leading to hyperpolarisation and (iii) reduced cyclic adenosine monophosphate

(cAMP) production via inhibition of adenylyl cyclase. Overall this results in reduced

neuronal cell excitability leading to a reduction in transmission of nerve impulses along

with inhibition of neurotransmitter release (Figure 7) (Hawes et al., 2000).

24

Figure 7. Schematic representation of intracellular responses to NOP receptor activation.

N/OFQ inhibits forskolin-stimulated cellular cAMP production. Forskolin is used

to directly activate adenylyl cyclase and consequently increase cAMP production.

Elevating cAMP via other receptor driven systems (e.g. via the D1 dopamine receptor) is

also inhibited by N/OFQ (Chan et al., 1998). Both the endogenous and recombinant NOP

receptors are capable of inhibiting the formation of cAMP with remarkably consistent EC50

values in a range of cell systems.

The peptide also inhibits several types of voltage-gated Ca2+ channels: for example

in human SH-SY5Y neuroblastoma cells it produces a partial inhibition of N-type Ca2+

conductance with an IC50 value of about 40 nM (Connor et al., 1996a), and in dissociated

rat hippocampal neurones the peptide partially inhibits the three major types of Ca2+

channels, L, N and P/Q (Knoflach et al., 1996). The inhibition is no longer seen after β

pertussis toxin treatment and cannot be prevented by high doses of naloxone. N/OFQ has

K+

β

cAMPATP

+-

-

Ca2+

GTP

GDP α

γ

Adenylate Cyclase

Hyperpolarisation of neurons Reduced neurotransmitter release

Reduced intracellular cAMP

Opioid

Receptor

25

been shown to mediate a pronounced inhibition of N-type calcium channels, whereas other

calcium channel subtypes were not affected.

N/OFQ has also been reported to increase the inwardly rectifying K+ conductance

in rat brain slices containing the dorsal raphe nucleus (Vaughan et al., 1996), the locus

coeruleus (Connor et al., 1996b), and the periaqueductal grey (Vaughan et al., 1997), in

hippocampal slices (Madamba et al., 1999) and cultured hippocampal neurones (Amano et

al., 2000).

Collectively, these data are consistent with the hypothesis that N/OFQ acts

primarily to reduce synaptic transmission and neuronal excitability in the nervous system

(Meunier, 1997). In the CNS, studies using synaptosomes and brain slices revealed that

N/OFQ inhibits the release of noradrenaline (NA), serotonine (5-HT), dopamine (DA),

acetylcholine (Ach), γ-aminobutyric acid (GABA), and glutamate (Schlicker et al., 2000).

In the peripheral nervous system, studies showed the general modulatory effects

(mostly inhibitory) of N/OFQ on neurotransmitter release from sympathetic,

parasympathetic and nonadrenergic-noncholinergic sensory endings. In the respiratory,

cardiovascular, genitourinary and gastrointestinal systems N/OFQ exerts inhibitory effects

(Giuliani et al., 2000). Several isolated tissues from different species have been shown to

be sensitive to N/OFQ. In particular, the electrically stimulated mouse and rat vas deferens

and the guinea pig ileum have been described and used extensively in opioid receptor

pharmacology: the guinea pig ileum, whose myenteric neuronal network contains mainly

MOP receptors (Paton, 1957) has been shown to respond to N/OFQ (Calo et al., 1997;

Calo et al., 1996; Zhang et al., 1997); the mouse vas deferens whose nerve terminals

contain mainly DOP receptors (Hughes et al., 1975) and the rat vas deferens, whose nerves

contain an uncharacterized opioid receptor (Lemaire et al., 1978), have also been reported

to be N/OFQ sensitive preparations (Berzetei-Gurske et al., 1996; Calo et al., 1997; Calo et

al., 1996; Nicholson et al., 1998; Zhang et al., 1997). The twitch response in the three

preparations is due to nerve activation and subsequent release of neurotransmitter since

they are blocked by tetradotoxin (TTX). The release of NA from the sympathetic nerves is

the major cause of the contractions of mouse and rat vas deferens, since they are blocked

by the α-1 adrenoceptor antagonist prazosin. NOP receptors appear to be localized in

sympathetic terminals since N/OFQ inhibits twitch evoked by electrical field stimulation,

but does not modify contractions to exogenous NA (Calo et al., 1996). Similar results were

obtained in the guinea pig ileum since atropine and TTX sensitive contractions derive from

the release of Ach from cholinergic terminals of the myenteric plexus without affecting

26

responses to exogenous Ach, thus demonstrating the prejunctional localization of the NOP

receptor. In the three tissues the inhibitory effect of N/OFQ is not influenced by naloxone

suggesting that classical opioid receptors are not targeted by the peptide.

A similar picture has been found regarding the inhibitory effects of N/OFQ on

sensory fibers on the guinea pig bronchus (Fischer et al., 1998; Rizzi et al., 1999b),

cholinergic contractions of human bronchus (Basso et al., 2005), renal pelvis and heart

(Giuliani et al., 1996; Giuliani et al., 1997a). The rat anococcygeus has also been described

as a preparation, in which N/OFQ produces a concentration-dependent inhibition of the

adrenergic motor response to electrical field stimulation, but does not affect the response to

exogenous NA. In addition, selective opioid ligands do not exert any effect on this

preparation, suggesting that in this preparation the NOP receptor occurs without the co-

presence of the classical opioid receptors (Ho et al., 2000). In all the preparations analysed

above, the N/OFQ-NOP receptor system displays a prejunctional inhibitory function, as do

classical opioid receptors.

1.7 Biological effects of N/OFQ

Due to the widespread distribution of N/OFQ and NOP receptor, this peptidergic

system is involved in a wide range of physiological responses with effects noted in the

nervous system (central and peripheral), the cardiovascular system, the airways, the

gastrointestinal tract and immune system. The role of this peptidergic system has been

explored intensely with the pharmacological and biological tools available, such as i)

antisense oligonucleotides targeting NOP receptor or ppN/OFQ gene, ii) antibodies

directed against N/OFQ, iii) transgenic mice in which the receptor or the peptide precursor

genes have been genetically eliminated, iv) available stable and highly potent antagonists.

Some of the more well-studied and noteworthy biological actions modulated by this system

will be described below.

Pain regulation

Since the identification of N/OFQ there has been intense interest in the role of this

peptide in pain processing. This is based on various factors, including the similarity of

distribution of receptor and peptide to classical opioids within the defined pain pathway

and the structural similarity to classical opioids. Application of N/OFQ has been shown to

cause hyperalgesia, allodynia and analgesia. These conflicting findings are confounded by

species and or strain differences in test animals, known to be fundamental in the

27

supraspinal effects of nociception (Mogil et al., 1999). However, the route of

administration and nociceptive paradigm under investigation are of paramount importance.

Supraspinal level

N/OFQ was shown to increase pain sensitivity in mice and rats in the two initial

studies of the functions of this peptide (hence the name nociceptin; (Meunier et al., 1995;

Reinscheid et al., 1995)). However, the hyperalgesic effect of N/OFQ was only seen after

intracerebroventricular (i.c.v.), but not after intrathecal (i.t.) administration. It has been

demonstrated that N/OFQ’s most prominent role in supraspinal pain modulation is a

“functional opioid antagonism” directed against many different opioid receptor agonists

(Mogil et al., 2001). Since behavioural testing in pain models, in particular i.c.v. and i.t.

injections, expose animals to acute stress, the apparent pronociceptive action seen in the

initial studies may thus be interpreted as the reversal of stress-induced antinociception

rather than as a genuine pronociceptive or hyperalgesic effect (Zeilhofer et al., 2003). I.c.v.

injection of N/OFQ was stressful, resulting in a release of central endogenous opioid

peptides with their effects subsequently reversed by the delivered dose of N/OFQ

(Lambert, 2008). The suggestion for an anti-opioid role of N/OFQ has since been

corroborated by results obtained in a variety of assays: indeed, it has been shown that

N/OFQ counteracts the analgesic effect of the endogenous opioids (Tian et al., 1997a; Tian

et al., 1997b) or that of exogenously applied morphine (Bertorelli et al., 1999; Calo et al.,

1998b; Grisel et al., 1996; Zhu et al., 1997) or that of selective opioid receptor agonists

(King et al., 1998). Worthy of mention is the fact that tolerance develops to the antiopioid

effects of N/OFQ (Lutfy et al., 1999).

Since the NOP receptor and classical opioid receptors largely share the same

transductional mechanisms, it is reasonable to speculate that their opposite effects on pain

threshold are due to distinct localisations of N/OFQ and opioid peptides and their

respective receptors on the neuronal networks involved in pain transmission at the

supraspinal level. Many studies demonstrate that the net effects of N/OFQ on nociception

at supraspinal sites strongly depend on the activation state (resting versus sensitized) of

pain controlling neuronal circuits (Zeilhofer et al., 2003).

It is possible that N/OFQ plays a role in the physiological modulation of pain

signals under normal or pathologic conditions. This question can be best answered through

the use of N/OFQ receptor antagonists, or transgenic mice lacking the NOP receptor gene

and with antisense oligonucletides. Indeed, the involvement of the NOP receptor in N/OFQ

28

effects on nociception is supported by the following evidence: i) the pronociceptive action

of N/OFQ is no longer present in NOP(-/-) mice (Nishi et al., 1997; Noda et al., 1998); ii)

antisense oligonucleotides targeting the NOP receptor prevent the effect of N/OFQ (Tian et

al., 1997b; Zhu et al., 1997); the pronociceptive effect of N/OFQ is reversed by NOP

selective antagonists: [Nphe1]N/OFQ(1-13)-NH2 (Calo et al., 2000a; Di Giannuario et al.,

2001; Rizzi et al., 2001b), UFP-101 (Calo et al., 2002b), J-113397 (Ozaki et al., 2000;

Yamamoto et al., 2001) and SB-612111 (Rizzi et al., 2007a; Zaratin et al., 2004).

Spinal level

Many lines of evidence indicate that the spinal cord is an equally important CNS

area for nociceptive processing and its modulation by N/OFQ and classical opioids.

Neurons and fiber networks containing N/OFQ mRNA and N/OFQ like immunoreactivity

have been located in the dorsal spinal cord (Lee et al., 1997; Mamiya et al., 1998; Meis et

al., 1998; Meunier et al., 1995), and endogenously released N/OFQ can be detected

following electrical field stimulation of the spinal cord (Lai et al., 2000).

The role of N/OFQ in modulating pain threshold in the spinal cord is controversial.

Although some studies reported that i.t. injection of N/OFQ produces

hyperalgesia/allodynia (Hara et al., 1997; Inoue et al., 1999) others found no effect (Grisel

et al., 1996; Reinscheid et al., 1995). Most of the studies, however demonstrated that i.t.

N/OFQ induces an antinociceptive effect similar to that evoked by classical opioid receptor

agonists (Candeletti et al., 2000b; Erb et al., 1997; Hao et al., 1998; Kamei et al., 1999;

King et al., 1997; Nazzaro et al., 2007; Wang et al., 1999; Xu et al., 1996). While

tolerance develops to the antinociceptive effect of i.t. N/OFQ upon repeated

administration, there is no cross tolerance with morphine, suggesting that different

receptors are involved in the actions of the two agents (Hao et al., 1997). Differences in

animal species or even in strains, as well as in N/OFQ doses used, may account for the

conflicting results reported with N/OFQ in the spinal cord. Worthy of mention is the work

of Inoue et al. (1999) showing the dose response curve to N/OFQ is bell-shaped: very low

doses of peptide (fmol range) cause hyperalgesia, while at higher doses (nmol range)

N/OFQ is antinociceptive and blocks the scratching, biting and licking induced by i.t.

substance P.

Extremely interesting is the latest study of Ko and colleagues (Ko et al., 2006) as

this is the first to document the inhibitory action of spinal N/OFQ in a primate species. The

behavioural study showed that i.t. administration of N/OFQ dose-dependently produced

29

antinociception in monkeys that was blocked by a NOP receptor antagonist, J-113397, but

not by naltrexone. These results provide the first functional evidence of spinal N/OFQ-

induced antinociception in primates and indicate that activation of spinal NOP receptors

may be a potential target for spinal analgesics.

Collectively, the cellular mechanisms of the pronociceptive effects of N/OFQ in the

spinal cord are still rather obscure, whereas inhibition of excitatory synaptic transmission

presents as a clearly defined cellular mechanism underlying the spinal analgesia. The

combination of opioid-like analgesia by N/OFQ at the level of the spinal cord with

functional opioid antagonism at supraspinal sites, where most of the unwanted effects of

classical opioids arise, has promoted the idea that NOP receptor agonists might be better

tolerated spinally acting analgesics. It should however be noted that the only well studied

non-peptide NOP receptor agonist Ro 64-6198 was anxiolytic, but not antinociceptive in

acute pain models (Jenck et al., 2000); Ro 64-6198 was recently reported to have an

antiallodynic effect mediated by NOP receptors in a neuropathic pain model (Obara et al.,

2005). Nevertheless, further studies with other NOP receptor agonists and in chronic pain

models are desirable.

Nociceptive responses to acute noxious heat in NOP(-/-), ppN/OFQ(-/-) and double

knockout mice were indistinguishable from those of NOP(+/+). However, NOP(-/-),

ppN/OFQ(-/-) and double knockout mice showed markedly stronger nociceptive response

during prolonged nociceptive stimulation (Depner et al., 2003). These results indicate that

the N/OFQ system contributes significantly to endogenous pain control during prolonged

nociceptive stimulation but does not affect acute pain sensitivity (Depner et al., 2003).

There have been attempts to correlate human plasma N/OFQ levels with pain but

the results of these studies have been contradictory. An increase in N/OFQ levels in acute

and chronic pain compared with controls was reported (Ko et al., 2002b). However, a

decrease in N/OFQ levels was noted in fibromyalgia syndrome (Anderberg et al., 1998)

and cluster headache (Ertsey et al., 2004).

Modulation of locomotor activity

One of the seminal investigations of N/OFQ reported a dose-dependent decrease in

locomotor activity (i.e., hypolocomotion) when the peptide was given supraspinally

(Reinscheid et al., 1995). This effect was significant only after i.c.v. application of 10 nmol

30

N/OFQ. This finding was later confirmed by other authors in mice (Nishi et al., 1997;

Noble et al., 1997; Noda et al., 1998).

Repeated daily N/OFQ injections result in rapid development of tolerance to this

depressor effect on locomotion behaviour (Devine et al., 1996). The action of N/OFQ is

insensitive to naloxone (Noble et al., 1997), while it is reversed by NalBzOH (Noda et al.,

1998). The locomotor-inhibiting effects of N/OFQ seen in NOP(+/+) animals were not

seen in the NOP(-/-) mice, confirming the involvement of the NOP receptor in the effect

(Nishi et al., 1997; Noda et al., 1998). However the spontaneous locomotor activity of

NOP(-/-) mice is not different from that displayed by NOP(+/+) littermates which suggest

that the N/OFQ-NOP receptor system does not play a tonic role in the physiological

regulation of locomotion. When microinjected directly into the hippocampus or

ventromedial hypothalamus, but not the nucleus accumbens, high doses of N/OFQ (10–25

nmol) significantly decrease locomotor activity (Sandin et al., 1997).

N/OFQ has also been reported to stimulate locomotor activity and exploratory

behaviour at very low doses (0.01-0.1 nmol) (Florin et al., 1996). This effect of N/OFQ has

been related to the anxiolitic-like actions of the peptide (Jenck et al., 1997). Thus, N/OFQ

shows a biphasic dose response curve for locomotor activity: stimulation at low doses

(0.01-0.1 nmol), inhibition at high doses (1-10 nmol).

It has been demonstrated that firing activity of dopaminergic cells of the substantia

nigra, which express NOP receptors, is inhibited by microinjection of N/OFQ (Marti et al.,

2004b). When microinjected into the substantia nigra, N/OFQ reduces dopamine release in

the striatum and locomotor activity (Marti et al., 2004b). Conversely, the NOP receptor

antagonists, UFP-101 and J-113397, injected into the substantia nigra, enhanced striatal

dopamine release and facilitated motor performance (Marti et al., 2004b). These data

confirm that improvement in locomotor activity is due to the enhanced striatal dopamine

release caused by blockade of endogenous N/OFQ signalling. The inhibitory role played

by endogenous N/OFQ on motor activity was additionally strengthened by the finding that

mice lacking the NOP receptor gene outperformed wild-type mice on the exercise

stimulated locomotion (rotarod test) (Marti et al., 2004b). Microinjection of UFP-101 into

the substantia nigra also reversed akinesia in haloperidol-treated (Marti et al., 2004a) or 6-

hydroxydopamine-hemilesioned rats (Marti et al., 2005). Enhancement of N/OFQ

expression and release was observed in the latter parkinsonism model (Marti et al., 2005).

Haloperidol-induced motor impairment and the dopaminergic neuronal toxicity induced by

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, but not methamphetamine, were partially

31

abolished in ppN/OFQ(-/-) mice (Brown et al., 2006; Marti et al., 2005). Increased

locomotor activity was observed in NOP(-/-) mice (Marti et al., 2004b) and in rats treated

with antisense-NOP (Blakley et al., 2004) or antisense-ppN/OFQ (Candeletti et al., 2000a).

These studies suggest that endogenous N/OFQ might have a negative regulation in striatal

dopamine levels and motor activity.

In the last few years, using a battery of behavioural tests, the group of prof. Morari

was first to show that NOP receptor antagonists like J-113397 attenuated parkinsonian-like

symptoms in 6-hydroxydopamine hemilesioned rats by reducing glutamate release in the

SN whereas deletion of the NOP receptor gene conferred mice partial protection from

haloperidol-induced motor depression (Marti et al., 2005), They subsequently showed that

coadministration of the NOP receptor antagonist J-113397 and L-DOPA to 6-

hydroxydopamine hemilesioned rats produced an additive attenuation of parkinsonism: J-

113397 and L-DOPA decreased thalamic GABA release and attenuated akinesia, their

combination resulting in a more profound effect (Marti et al., 2007). Very recently, it has

been demonstrated that Trap-101, a non-peptide NOP antagonist, changes motor activity in

naive rats and mice and alleviates parkinsonism in 6-hydroxydopamine hemilesioned rats:

Trap-101 stimulates motor activity at 10 mg/kg and inhibits it at 30 mg/kg (Marti et al.,

2008); such dual action was observed in NOP(+/+) but not in NOP(-/-) mice suggesting a

specific involvement of NOP receptors (Marti et al., 2008). Overall, the present study

provides novel insights into the mechanisms underlying the antiparkinsonian action of

NOP receptor antagonists that may be used alone or as an adjunct to L-DOPA in the

therapy of Parkinson’s disease. This indication has also been confirmed in non-human

primates (Viaro et al., 2008; Visanji et al., 2008).

Anxiolytic-like action

Different laboratories have reported anxiolytic-like effects in response to

intracerebroventricular administration of N/OFQ in rodents in several models of anxiety.

The peptide and its receptor are found in a number of central nervous system loci

involved in emotion and stress regulation, including the amygdala, septal region, locus

coeruleus, and hypothalamus (Gavioli et al., 2006).

A number of standard behavioural assays reveal the ability of supraspinal N/OFQ to

block fear and anxiety in both rats and mice (Jenck et al., 1997). Interestingly, these effects

of N/OFQ were evident at relatively low doses (<1 nmol), which do not modify animal

gross behaviour and are inactive with regard to other functions (i.e., nociception, food

32

intake, etc.). Later, other laboratories confirmed the anxiolytic-like effects of the natural

peptide N/OFQ in mice in the elevated plus-maze test (Gavioli et al., 2002), in the

holeboard test (Kamei et al., 2004), and in the defence test battery (Griebel et al., 1999;

Vitale et al., 2006). Furthermore, at relatively low doses, several non-peptide agonists from

Roche (Ro 65-6570 and Ro 64-6198) are generally reported as anxiolytic (Varty et al.,

2005; Wichmann et al., 1999). After peripheral administration in the range of doses 0.1–3

mg/kg, Ro 64-6198 promoted anxiolytic-like effects in rats in the elevated plus-maze, fear-

potentiated startle, and operant conflict tests (Jenck et al., 2000). This hypothesis has been

corroborated by the findings that genetically N/OFQ precursor deficient mice display an

increased susceptibility to acute and repeated stress, as compared to their wild-type

littermates (Koster et al., 1999). In a recent study from Schering-Plough the non-peptide

agonist SCH 221510 was shown to be anxiolytic but with a reduced side-effect profile

when compared with benzodiazepines (Varty et al., 2008). Pfizer has recently reported two

new non-peptide NOP receptor agonists: PCPB (Hirao et al., 2008a) and MCOPPB

(Hayashi et al., 2009; Hirao et al., 2008b); both orally active compounds showed

anxiolytic-like effect in mice.

Importantly, very little information is present in the literature regarding the effects

of selective NOP receptor antagonists on anxiety. It has been reported that the non-peptide

molecule J-113397 at 10 mg/kg i.p. antagonized the anxiolytic-like effect of Ro 64-6198 in

the conditioned lick suppression test in rats without having any effect in this assay per se

(Varty et al., 2005). Although very few studies have been performed to date on this topic,

the available evidence obtained with two chemically unrelated NOP antagonists, i.e., J-

113397 and UFP-101 (Gavioli et al., 2006; Vitale et al., 2006), suggest that the acute

blockade of NOP receptor does not modify the level of anxiety in rodents. In other words,

these results suggest that N/OFQergic signalling does not tonically control anxiety-related

behaviour.

Surprisingly, Fernandez et al. (2004) observed anxiogenic-like effects of N/OFQ

given by i.c.v. injection in several anxiety-related procedures (i.e., open field, elevated plus

maze, and dark-light preference) in rats. Explanations given by the authors are a difference

in baseline stress between studies, and/or strain differences.

Even though a control experiment suggested that the anxiogenic-like effects were,

at least in part, independent of effects on locomotion (Fernandez et al., 2004), a recent

study by Vitale et al. (2006) may suggest the opposite. They replicated the anxiogenic-like

effects of N/OFQ in the rat elevated plus-maze test, but observed anxiolytic-like effects

33

after 2 subsequent administrations of N/OFQ (Vitale et al., 2006). This change from

anxiogenic- to anxiolytic-like effect of N/OFQ was accompanied by tolerance to the

hypolocomotor effects of N/OFQ, suggesting that the anxiolytic-like effects of acute

N/OFQ were masked by hypolocomotor effects.

So far the anxiolytic mechanisms of N/OFQ are not well understood. It is likely that

N/OFQ effects on anxiety may depend on the ability of this peptidergic system to modulate

endogenous 5-HTergic pathways since 5-HT is considered to play a pivotal role in the

control of anxiety and fear (Millan, 2003). Moreover, recent findings suggested that the

anxiolytic-like effects of N/OFQ might be mediated via activation of the

GABA/benzodiazepine system and the effects of N/OFQ may relate to the regulation of

anxiety states in the amygdala (Gavioli et al., 2008; Uchiyama et al., 2008).

Several lines of evidence also suggest that endogenous N/OFQ has an important

role in anxiety and stress regulation. Enhanced anxiety was shown in ppN/OFQ(-/-) mice

(Kest et al., 2001; Ouagazzal et al., 2003; Reinscheid et al., 2002) and antisense-NOP

treated rats (Blakley et al., 2004), but not in NOP(-/-) mice (Mamiya et al., 1998). Gavioli

et al. (2007) demonstrated that there are no clear differences between NOP(-/-) and

NOP(+/+) mice in some classical models of anxiety (open-field, hole-board and marble-

burying tests). In contrast, when subjected to other models of anxiety such as novelty-

suppressed feeding behaviour and the elevated T-maze test, NOP(-/-) mice display lower

anxiety-related behaviours compared to NOP(+/+) mice (Gavioli et al., 2007). In the

elevated plus-maze and light-dark box, NOP(-/-) mice displayed increased anxiety-related

behaviour (Gavioli et al., 2007).

Interestingly, the impact of ppN/OFQ deletion on the anxiety-like behaviours is

more significant in group-housed, as compared with individual-housed mice, and male

mice were more susceptible than females (Ouagazzal et al., 2003). In mice, differences in

anxiety states are associated with differences in G protein coupling efficiency in the

nucleus accumbens (but not in 12 other brain regions) (Le Maitre et al., 2006). A likely

explanation of this finding is that the observed increase in coupling in non-anxious mice

leads to increased N/OFQ-mediated transmission and thus protects from anxiety (Le

Maitre et al., 2006).

It has also been suggested that N/OFQ can act as a functional corticotrophin

releasing factor (CRF) antagonist, since it is able to revert the hypophagia induced by

either stress or the central administration of CRF (Ciccocioppo et al., 2001). Since CRF is

a major mediator of stress and a potent anxiogenic agent, the functional relationships

34

between the N/OFQ and CRF systems are worthy of further investigation aimed at

clarifying the mechanisms by which N/OFQ exerts its anxiolytic-like effects.

However, the information that has been available to date has been too limited to

propose a mechanistic interpretation of the anxiolytic-like effects of N/OFQ. Other studies

aimed to the identification of the brain areas involved in this action are needed for a better

understand N/OFQ role in this field.

Antidepressant-like action

Studies performed in rodents subjected to behavioural despair tests support a role of

the N/OFQ-NOP receptor system in the modulation of mood behaviours.

NOP receptor antagonists, including [Nphe1]N/OFQ(1-13)-NH2, J-113397, UFP-

101 and SB-612111 reduced immobility time in both the forced swim and tail suspension

tests. N/OFQ (i.c.v.) alone did not affect immobility time (Gavioli et al., 2004; Redrobe et

al., 2000; Rizzi et al., 2007a).

In our laboratories, using a combined pharmacological and genetic approach, we

demonstrated that blockade of N/OFQ-NOP receptor signalling in the brain produces

antidepressant-like effects in the mouse and rat forced swimming test and in the mouse tail

suspension test. I.c.v. injection of N/OFQ did not induce any behavioural modification in

mice, but the co-administration of 1 nmol of N/OFQ reversed the antidepressant-like effect

induced by the NOP receptor antagonists UFP-101 (Gavioli et al., 2003; Gavioli et al.,

2004). In addition, N/OFQ (1 nmol) also reverted the effects induced by the non-peptide

NOP receptor antagonist J-113397 in the mouse forced swimming test (Gavioli et al.,

2006). Whereas the immobility time in NOP(-/-) mice is less than that in NOP(+/+), the

antidepressant-like effects of NOP receptor antagonists were not observed in NOP(-/-)

mice (Gavioli et al., 2003), suggesting that endogenous N/OFQ plays a role in those

depression-like behaviours. Treatment with UFP-101 (10 nmol) reduced immobility time

in NOP(+/+) mice, while it was inactive in mice lacking the NOP receptor (Gavioli et al.,

2003). Systemic administration of J-113397 (20 mg/kg) promoted a statistically significant

reduction in immobility time in the forced swimming test in NOP(+/+), but not in NOP(-/-)

animals. Additionally, SB-612111 (10 mg/kg, i.p.) reduced the immobility time in

NOP(+/+) mice while being inactive in NOP(-/-) animals (Rizzi et al., 2007a).

Moreover, it has been demonstrated that antidepressant-like effects elicited by the

selective NOP receptor antagonist UFP-101 are probably due to the block of the inhibitory

35

effects of endogenous N/OFQ on brain monoaminergic (in particular serotonergic)

neurotransmission (Gavioli et al., 2004).

Vitale and colleagues investigated the effect of UFP-101 in the chronic mild stress

paradigm in rats; UFP-101 (10 nmol/rat, i.c.v. continuously infused by means of

minipumps for 24 days) did not influence sucrose intake in non stressed animals, but

reinstated the basal sucrose consumption in stressed animals, beginning from the second

week of treatment as did fluoxetine (10 mg/kg, i.p.), used as reference drug (Vitale et al.,

2008).

There is just one human study (Gu et al., 2003) in which plasma N/OFQ level was

elevated in post-partum depressive women. This limited small study agrees with the notion

that post-partum depression results from reduced 5-HT levels and that this is accompanied

by elevated N/OFQ with the increase in N/OFQ possibly causing the fall in 5-HT

(Lambert, 2008). Thus, NOP receptor antagonists may have the potential to be novel

antidepressants.

Overall, these pharmacological and genetic findings suggest that the blockade of

N/OFQ signaling induces antidepressant-like effects in rodents. Importantly, all these

observations were performed after acute drug administration, and no data are currently

available subsequent to chronic drug exposure. Thus, further studies aimed at the

evaluation of the antidepressant-like effects of NOP receptor antagonists in animal models

based on chronic stress may generate important information about the role of this

peptidergic system in mood disorders.

Food intake

Soon after the isolation of N/OFQ, Pomonis and colleagues (Pomonis et al., 1996)

showed that supraspinal N/OFQ (1–10 nmol) increased food intake in the satiated rat.

N/OFQ effects are short-lasting, specific to food intake with neither water intake nor 1%

sucrose intake affected, and accompanied by transient hypolocomotion (Polidori et al.,

2000a).

N/OFQ hyperphagia can be blocked by antisense treatment to NOP mRNA

(Leventhal et al., 1998), competitive NOP antagonism (Polidori et al., 2000a) and

functional antagonism by nocistatin (Olszewski et al., 2000). Surprisingly,

naloxone/naltrexone pretreatment also blocks N/OFQ effects on food intake (Leventhal et

al., 1998; Pomonis et al., 1996), although this is probably due to classical opioid receptors

being involved in feeding control at a distal site or affecting motivational processes related

36

to food intake. In addition, it was shown that the orexigenic action of 1 nmol of N/OFQ

was prevented by SB-612111 (1 mg/kg) and no longer evident in NOP(-/-) animals,

indicating that the orexigenic effects induced by N/OFQ are exclusively due to NOP

receptor activation (Rizzi et al., 2007a).

The orexigenic action of N/OFQ is suggested to be attributed to both the inhibition

of anorexigenic systems and the activation of orexigenic systems (Olszewski et al., 2004).

N/OFQ has been found to inhibit pathways that promote termination of food intake in the

hypothalamic satiety centers, such as oxytocinergic neurons in the paraventricular nucleus

and neurons in the arcuate nucleus (Olszewski et al., 2004).

Moreover, Ciccocioppo et al. (2004) found that N/OFQ, at doses without

hyperphagic effects, inhibited stress-induced anorexia and that this anti-anorexic effect is

due to the fact that N/OFQ acts as a functional antagonist of corticotrophin-releasing factor

(CRF) at the bed nucleus of the stria terminalis (Ciccocioppo et al., 2004).

[Nphe1]N/OFQ(1-13)-NH2 did not affect food consumption per se in satiated rats, but

reduced that in food-deprived rats (Polidori et al., 2000a). UFP-101 also did not affect free

feeding in the rat (Economidou et al., 2006b). This suggested that endogenous N/OFQ

plays a role in orexigenic tone in response to food deprivation but not in normal feeding.

On the contrary, Rizzi et al. (2007a) showed that the antagonist SB-612111 (1 and 10

mg/kg, i.p.), tested in food deprived mice, did not modify food intake. Thus, the data

obtained with SB-616211 suggest that in mice, unlike rats (Polidori et al., 2000a), the

N/OFQ-NOP receptor system does not play a major role in controlling food intake induced

by food deprivation.

All these studies indicate that the hyperphagic and the anti-anorectic effect of

N/OFQ are mediated by separate brain structures and that synthetic N/OFQ agonists might

have therapeutic potential as orexigenic drugs (Ciccocioppo et al., 2004; Economidou et

al., 2006b).

Reward and addiction

In animal models aimed at elucidating the rewarding properties of drugs of abuse

the conditioned place preference (CPP) test is commonly used. In this assay N/OFQ has

been shown to reduce CPP to alcohol (Ciccocioppo et al., 1999; Kuzmin et al., 2003),

amphetamines (Kotlinska et al., 2003), cocaine (Kotlinska et al., 2003; Sakoori et al.,

2004), and morphine (Sakoori et al., 2004) indicating that this peptide was reducing reward

to these stimuli. N/OFQ alone was inactive. All these experiments measured the

37

acquisition or reinstatement of drug preferences, either as a conditioned response (place

preference) or self-administration of the drug itself. However, it should be mentioned that

N/OFQ failed to block heroin self-administration (Walker et al., 1998). Another study also

showed that N/OFQ was effective in preventing stress-induced alcohol-seeking behaviour

but not cocaine-seeking behaviour (Martin-Fardon et al., 2000). Finally, one study

demonstrated that N/OFQ was able to block sensitization to cocaine, independent of

context (Lutfy et al., 2002).

Since the mesolimbic dopaminergic system plays a pivotal role in opioid rewarding

properties (Wise, 1989), it has been suggested that N/OFQ attenuates conditioned place

preference to any type of drug of abuse by inhibiting its stimulatory effect on mesolimbic

dopamine release from the nucleus accumbens (Murphy et al., 1999). In fact, i.c.v. N/OFQ

effectively inhibits dopamine release (as evaluated by in vivo microdialysis) in the nucleus

accumbens of the rat stimulated by systemically injected morphine (Di Giannuario et al.,

1999). Alternatively, the inhibitory effects of N/OFQ could be explained by the finding

that N/OFQ inhibits GABAergic transmission and blocks ethanol-induced increase of

GABA release in the central amygdala (Roberto et al., 2006). Interestingly, Ciccocioppo et

al. (2007) found that buprenorphine, a partial agonist at MOP and NOP receptors,

increased alcohol intake at lower doses through MOP receptors while decreased it at higher

doses through NOP receptors. It is suggested that the therapeutic potential of

buprenorphine in drug addiction might be attributed to its NOP receptor activation, but not

to its activation of classical opioid receptors.

Recent findings demonstrated that the psychostimulant and rewarding actions of

buprenorphine were enhanced in NOP(-/-) mice as compared to their NOP(+/+) littermates.

However, these actions of morphine were not altered in mutant mice. Buprenorphine

displaced specific binding of [3H]-N/OFQ in brain homogenates of NOP(+/+) mice;

together these results suggest that the ability of buprenorphine to interact with NOP

receptor compromises its acute motor stimulatory and rewarding actions (Marquez et al.,

2008).

Even though more work is obviously necessary to fully understand the effects of

N/OFQ on drug reward and the mesolimbic dopamine system, it appears that N/OFQ

agonists might provide useful compounds to control the rewarding aspects of drugs.

38

Learning and memory

N/OFQ may play a role in memory and learning processes since there is a high

density of NOP receptors in the anterior cingulate, frontal cortex, basolateral complex of

the amygdala and hippocampus. In fact N/OFQ injected into the hippocampus impairs

spatial learning (Sandin et al., 1997) and in vitro it inhibits synaptic transmission and long-

term potentiation in rat hippocampal slices (Yu et al., 1997). Recently it was also seen that

endogenously released N/OFQ interacts with noradrenergic activity within the basolateral

complex of the amygdala in modulating memory consolidation (Roozendaal et al., 2007).

In line with these findings, NOP(-/-) mice show greater learning ability and have

better memory retention than wild-type control mice (Manabe et al., 1998). The

impairment of learning induced by N/OFQ can be reversed by nocistatin (Hiramatsu et al.,

1999b) or by the non-selective NOP receptor antagonist NalBzOH (Mamiya et al., 1999).

Moreover, a peptidic NOP receptor antagonist Ret-Noc-OMe, has been reported to

strengthen memory retention in a passive avoidance test in mice (Jinsmaa et al., 2000).

It is worthy of note that pre-treatment with [Nphe1]N/OFQ(1-13)-NH2, a NOP

receptor antagonist, prevented NOP-induced deficits. Using a pure pharmacological

approach, i.e. NOP receptor blockade, a role for the N/OFQ and its receptor in learning and

memory has been demonstrated (Redrobe et al., 2000).

Very recent study showed that intracerebroventricular or intrahippocampal

infusions of N/OFQ impair long-term memory formation in the mouse object recognition

task. The synthetic NOP receptor agonist Ro 64-6198, administered systemically, also

produced amnesic effects that were blocked by coinfusion of the NOP receptor antagonist

UFP-101, into the dorsal hippocampus. In contrast, Ro 64-6198 had no effect on short-term

memory or recall performances (Goeldner et al., 2008). Immunoblotting analysis revealed

a strong suppressive action of Ro 64-6198 on learning-induced upregulation of

hippocampal extracellular signal-regulated kinase (ERK) phosphorylation, which is crucial

for long-term information storage (Goeldner et al., 2008). Thus, N/OFQ-NOP receptor

system negatively regulates long-term recognition memory formation through a

hippocampal ERK signalling mechanism (Goeldner et al., 2008).

Collectively, these findings suggest that the N/OFQ-NOP receptor system may play

negative roles in learning and memory, and that NOP receptor antagonists might be worthy

of testing as drugs for memory disorders.

39

Effects in the gastrointestinal tract

Like morphine or other opioid receptor agonists, N/OFQ inhibits in vitro

neurogenic contractions of the stomach and the small intestine in a variety of species,

including guinea pigs (Calo et al., 1996; Zhang et al., 1997), pigs (Osinski et al., 1999b),

rats (Yazdani et al., 1999) and rabbits (Pheng et al., 2000). This depressor effect is

resistant to blockade by naloxone. In contrast, N/OFQ causes concentration-dependent

contractions in proximal rat colon, without changes in stomach, jejunum or ileum

(Taniguchi et al., 1998; Yazdani et al., 1999). N/OFQ also contracts proximal and distal

segments of mouse colon (Menzies et al., 1999; Osinski et al., 1999a; Rizzi et al., 1999a).

In vivo, similarly to opioids, central administration of N/OFQ also inhibits colon

transit in the mouse (Osinski et al., 1999a). On the other hand, Taniguchi and collegues

(Taniguchi et al., 1998) reported that N/OFQ administered subcutaneously in rats actually

accelerated transit rate in the large intestine, an action opposite to that induced by

morphine or selective opioid receptor agonists. Broccardo and colleagues demonstrated

that the NOP receptor antagonist [Nphe1]N/OFQ(1-13)-NH2 blocked the N/OFQ-evocked

gastrointestinal anti-transit effect (Broccardo et al., 2004). It is worthy of note that

[Nphe1]N/OFQ(1-13)-NH2 per se stimulated gastric acid secretion.

Distal colonic contractions induced by N/OFQ were also dose-dependently

antagonized by the NOP non-peptide antagonist J-113397 that behaves as selective NOP

antagonist in the rat colon (Tada et al., 2002).

All these findings led suggest that the N/OFQ-NOP receptor system is

pharmacologically distinct from opioids but functionally very similar, and could represent

a new target for the development of drugs (NOP receptor agonists) to reduce intestinal

motility.

In addition to the well-characterized inhibition of gastric motility, recent studies

demonstrated that N/OFQ increases gastric mucosal resistance to ethanol induced lesions

by operating both in the central nervous system and in the periphery (Morini et al., 2005).

This effect is mediated by the NOP receptor since the selective N/OFQ antagonist UFP-

101 completely prevents the protective effects of N/OFQ (Morini et al., 2005). There is

evidence for central and peripheral components to the regulation of gastrointestinal

function: vagal cholinergic and sympathetic pathways mediate the central activity of

N/OFQ, whereas vagal non-muscarinic pathways mediate the peripheral activity of the

peptide (Broccardo et al., 2004; Ishihara et al., 2002).

40

An elegant study recently showed that N/OFQ and UFP-112, a novel highly potent

NOP agonist, when administered intracerebroventrically and intraperitoneally decreased

bead expulsion time and reduce the percentage of rats with castor oil-induced diarrhoea;

UFP-112 showed greater efficacy, higher potency and longer-lasting effects than N/OFQ

(Broccardo et al., 2008). These findings indicate that, in the rat, the central and peripheral

N/OFQ system has an inhibitory role in modulating distal colonic propulsive motility

under physiological and pathological conditions (Broccardo et al., 2008).

Further studies are necessary to investigate the mechanism(s) accounting for this

interesting effect of N/OFQ.

Effects in the airways

N/OFQ was found to inhibit contractions of the guinea pig isolated bronchus

(Fischer et al., 1998; Rizzi et al., 1999b) of the rat isolated trachea and bronchus (Wu et

al., 2000) induced by electrical field stimulation and of the cholinergic contractions of

human bronchus (Basso et al., 2005).

N/OFQ inhibited the cough responses provoked by capsaicin in guinea pigs or by

mechanical stimulation of intrathoracic airways in cats (Bolser et al., 2001; McLeod et al.,

2001). These antitussive actions might be mediated at both central and peripheral sites.

N/OFQ decreased capsaicin-induced Ca2+ influx in nodose ganglia (McLeod et al.,

2004), the sensory ganglia involved in the cough reflex (Reynolds et al., 2004), and the

airway contraction in a manner blocked by tertiapin, an inwardly rectifying K+ channel

blocker (Jia et al., 2002). In the brain, there are dense NOP receptors in the medullar

nucleus tractus solitarius (Judd et al., 1996), which provides polysynaptic inputs to second-

order neurons that modulate respiratory neuron activities (Reynolds et al., 2004).

This antitussive effect can be mimicked by the non-peptide agonist Ro 64-6198 in a

J-113397-sensitive manner (McLeod et al., 2004). Codeine is the current gold-standard

antitussive agent but has a poor side-effect profile that is typical of MOP opioid receptor

agonists (such as nausea, constipation, tolerance and dependence). So, orally active NOP

agonists might represent a viable alternative for the treatment of cough (Lambert, 2008).

Cardiovascular system

When given intravenously (i.v.) in anaesthetised rats, N/OFQ induces transient

hypotension and bradycardia (Champion et al., 1997; Giuliani et al., 1997b). Similar

results have been obtained in conscious rats (Kapusta et al., 1997) and mice (Madeddu et

41

al., 1999), indicating that anaesthesia does not affect the cardiovascular effects of N/OFQ

and that these effects are not restricted to the rat. Interestingly, N/OFQ induces similar

cardiovascular effects when injected i.c.v. (Kapusta et al., 1997) or into the rostral

ventrolateral medulla of the rat (Chu et al., 1999). The effects occur at both central and

peripheral sites. The most compelling evidence for peripheral effects is in the hypotension

and bradycardia produced by intravenous administration of N/OFQ, a peptide that does not

cross the blood-brain barrier (Lambert, 2008). It has been suggested that as the

sympatholytic guanethidine reduced the hypotensive effects of N/OFQ, then this peptide

acts to inhibit sympathetic control of the cardiovascular system (Giuliani et al., 1997b). In

addition, the bradycardic effects of N/OFQ were reduced by vagotomy, indicating that

N/OFQ increased parasympathetic activity (Giuliani et al., 1997b).

On the other hand, N/OFQ has been shown to increase blood pressure and heart rate

in sheep following i.v. administration (Arndt et al., 1999). Thus, there may be important

differences in the cardiovascular effects of N/OFQ in different species.

N/OFQ also produces vasodilation in several isolated arteries of the cat (Gumusel et

al., 1997) and in pial arteries of the pig (Armstead, 1999) and in the mesenteric resistance

arteries of the rat (Champion et al., 1998). These studies also demonstrated that the

vasodilator responses to N/OFQ were not prevented by naloxone, nitric oxide synthase

inhibitors, atropine, phentolamine or by a CGRP receptor antagonist. Recently a study

showed that in a rat mesenteric microcirculation model, intravenous administration of

N/OFQ dilated arterioles and venules (Brookes et al., 2007); dilation of these non-

innervated vessels was blocked by histamine antagonists and mast-cell stabilizers,

suggesting that the N/OFQ-mediated dilation of the microcirculation is secondary to mast-

cell release of histamine.

Renal function

The i.v. infusion of N/OFQ produces a marked increase in urine flow rate and a

decrease in urinary sodium excretion (Kapusta et al., 1997). Concurrent with diuresis,

N/OFQ infusion produced hypotension with no change in heart rate; this is in contrast to

the concurrent bradycardia and hypotension elicited by N/OFQ when this peptide is

administered as an i.v. bolus (Bigoni et al., 1999; Champion et al., 1997; Giuliani et al.,

1997b; Madeddu et al., 1999), intrathecally (Lai et al., 2000), or when microinjected into

the lateral cerebroventricle (Kapusta et al., 1999a; Kapusta et al., 1999b; Kapusta et al.,

1997; Shirasaka et al., 1999). Low doses of N/OFQ (i.v. infusion) also tended to decrease

42

urinary sodium excretion without changes in heart rate or mean arterial pressure. These

findings also suggest that the i.v. infusion lower doses of N/OFQ can be used to separate

the cardiovascular and renal responses produced by this compound, with the peptide

having a more pronounced effect on the renal handling of water (Kapusta, 2000).

Following i.c.v. microinjection, N/OFQ produced a marked diuresis, antinatriuresis and

renal sympathoinhibition in conscious rats (Kapusta et al., 1999a; Kapusta et al., 1999b;

Kapusta et al., 1997; Shirasaka et al., 1999)

It is worthy of note that a class of NOP ligands, the partial agonists (i.e. Ac-

RYYRWK-NH2, Ac-RYYRIK-NH2 (Dooley et al., 1997), [F/G]N/OFQ(1-13)-NH2

(Guerrini et al., 1997), ZP120 (Larsen et al., 2001) have been shown to behave as full

agonists on cardiovascular and renal functions, mimicking the effects of N/OFQ, when

given i.c.v. (Kapusta et al., 1999b). In contrast, the i.v. bolus injection of the same NOP

receptor partial agonists produced responses unlike N/OFQ; N/OFQ evoking profound

bradycardia and hypotension with no change in urine output, and i.v. bolus NOP receptor

partial agonists eliciting water diuresis without altering cardiovascular function (Kapusta et

al., 2005a; Kapusta et al., 2005b). Indeed, Kapusta et al. showed that ZP120 (i.v. bolus or

i.v.infusion) produced, in rats, a sodium-potassium-sparing aquaresis and a mild

vasodilatory response without reflex tachycardia (Kapusta et al., 2005b).

Activation of NOP receptors in the paraventricular nucleus (PVN) of the

hypothalamus by N/OFQ produces bradycardia, renal sympathoinhibition, and water

diuresis. Recently, the group of Kapusta (Krowicki et al., 2006) showed that endogenous

N/OFQ produces a tonic inhibition on PVN activity since UFP-101, when injected into the

PVN, increased heart rate and renal sympathetic nerve activity and decreased urine flow

rate.

In summary, it was seen that in conscious rats NOP receptor partial agonists

produced functionally selective effects on cardiovascular and renal function ranging from

full agonist (i.c.v., cardiovascular depressor; i.c.v. and i.v., water diuresis), partial agonist

(i.v., submaximal hypotension without altering heart rate) to antagonist (i.v., blockade of

N/OFQ-evoked bradycardia and hypotension) behaviour. Based on their ability to produce

a selective water diuresis after i.v. bolus injection without apparent adverse cardiovascular

or CNS effects, it can be proposed that metabolically stable NOP receptor partial agonists

(e.g., ZP120; (Kapusta et al., 2005b)) may be useful therapeutically as novel peripherally

acting aquaretics for the acute management of severe water retention and/or hyponatremia;

43

ZP120 was, hence, filed for phase II clinical trials for the treatment of acute

decompensated heart failure.

Micturition reflex

In anaesthetised rats, i.v. N/OFQ produced a dose-dependent suppression of the

micturition reflex induced by bladder distension or by topical application of capsaicin

(Giuliani et al., 1998). Similar results were obtained by administering the peptide i.c.v. or

i.t. indicating that N/OFQ inhibits the micturition reflex by acting at peripheral, spinal and

supraspinal sites (Lecci et al., 2000).

All these effects are not affected by naloxone, thus excluding the involvement of

opioid receptors. These animal studies were later confirmed in clinical studies. Indeed, the

urodynamic and clinical effects of N/OFQ were evaluated in normal subjects and in

patients with neurogenic bladder. A preliminary report (Lazzeri et al., 2001) and a

subsequent randomized, placebo controlled, double-blind study (Lazzeri et al., 2003)

demonstrated that intravesical instillation of 1 μM N/OFQ solution produce an inhibitory

effect on micturition reflex in selected groups of patients suffering from neurogenic

incontinence but not in normal subjects. These effects of N/OFQ are due to its ability to

selectively activate the NOP receptor as suggested by the fact that [desPhe1]N/OFQ, a

N/OFQ metabolite which does not bind NOP receptor, is inactive in these patients (Lazzeri

et al., 2003). Moreover, a recent study (Lazzeri et al., 2006) demonstrated that a daily

treatment with 1 mg N/OFQ intravesically for 10 days, but not the placebo, inhibited the

micturition reflex in patients suffering from neurogenic incontinence, thus demonstrating

the clinical efficacy of a prolonged NOP receptor agonist treatment. Based on these

findings N/OFQ selective and potent peptide agonists with long lasting effects in vivo may

be proposed as innovative drugs for treating patients suffering from neurogenic

incontinence.

Immune system

NOP receptors and N/OFQ are widely distributed throughout the immune system.

NOP receptor mRNA and protein have been found in a variety of immune cells including

mouse lymphocytes (Halford et al., 1995), human peripheral blood mononuclear cells

(Wick et al., 1995) and human circulating granulocytes, lymphocytes and monocytes (Fiset

et al., 2003; Peluso et al., 1998). Neutrophils are thought to be a source of N/OFQ in

inflammatory tissues (Fiset et al., 2003). N/OFQ can function as an immunosuppressant by

44

suppressing antibody production in mouse lymphocytes, by decreasing proliferation of

phytohemagglutinin-stimulated PBMCs, and by inhibiting mast cell function (Civelli,

2008). In addition, it was shown that N/OFQ stimulates human monocyte chemotaxis via

NOP receptor activation (Trombella et al., 2005). The importance of the N/OFQ system in

the immune response, however, remains to be clearly defined.

1.8 Pharmacology of NOP receptors

From the numerous modulatory actions of N/OFQ in several pathways, it is clear

that NOP may represent an important molecular target for the development of novel

therapeutics for several pathological conditions. The identification of new molecules

possibly of non-peptide nature that selectively activate (agonists) or block (antagonists) the

NOP receptor will represent a major achievement in this research field, providing

pharmacological tools for clarification of the physiological and pathophysiological roles of

this new system and ultimately for the identification of possible therapeutic agents acting

at the NOP receptor.

Peptide compounds usually show high selectivity and specificity but metabolic

instability and limited distribution. In contrast, non-peptides demonstrate better

pharmacokinetic features while their specificity is often low. There is an evident interest of

academia and pharmaceutical companies in developing both agonist and antagonist ligands

for the NOP receptor as potential drugs for various human disorders (see series of patents

quoted by Zaveri (2003) and (Bignan et al. (2005)).

Peptide ligands

N/OFQ shows a significant homology with dynorphin A. The first four amino acids

differ from the canonical opioid sequence only by the presence of Phe1 instead of Tyr1.

This difference may be sufficient to prevent N/OFQ binding to opioid receptors. In fact,

replacement of Phe1 by Tyr1 results in a peptide that also binds the opioid receptors (Calo

et al., 1997; Varani et al., 1999). Amidation of the C-terminus (N/OFQ-NH2) maintains

full potency and activity (Guerrini et al., 1997). Early studies to determine the minimum

active sequence showed that up to four C-terminal amino acids can be deleted without loss

of activity. Although the free acid N/OFQ(1–13)-OH loses receptor affinity, amidation of

the C-terminus to give N/OFQ(1–13)-NH2 restores potency and agonist activity

comparable to the parent peptide (Calo et al., 1996; Dooley et al., 1996; Reinscheid et al.,

1996). C-terminal amidation protects from degradation by carboxypeptidases and is now a

45

standard feature of most N/OFQ-based peptide ligands. In fact, the truncated peptide

N/OFQ(1–13)-NH2 has been used as a chemical template for SAR studies aimed at

investigation of novel ligands for the NOP receptor.

Initial structure-activity studies on N/OFQ(1–13)-NH2 by Guerrini et al. (1997)

determined that the N-terminal peptide FGGF is essential for activity and that Phe4 and

Phe1 appear to be important for receptor activation. Further studies on N-terminal

modification resulted in the discovery of a purported NOP antagonist in which the Phe1-

Gly2 amide bond was replaced with a pseudopeptide (CH2-NH) bond (Calo et al., 1998a;

Guerrini et al., 1998). This peptide [Phe1Ψ(CH2-NH)Gly2]N/OFQ(1–13)-NH2 abbreviated

as [F/G]N/OFQ(1–13)-NH2, was shown to behave as a selective and competitive

antagonist in the electrically stimulated guinea pig ileum and mouse vas deferens (Guerrini

et al., 1998). This report initiated a surge of in vitro and in vivo studies (Calo et al., 2000a)

which showed that this peptide behaved as an antagonist, partial agonist, or even full

agonist, depending on the tissue preparation. Thus, while it showed different levels of

partial agonist activity in [35S]GTPγS assays in CHO cells transfected with human or

mouse NOP (Berger et al., 2000b; Burnside et al., 2000), it showed full agonist activity in

several in vivo CNS assays (Calo et al., 1998b; Carpenter et al., 1998; Grisel et al., 1998;

Xu et al., 1998). McDonald and colleagues demonstrated that agonism is primarily

dependent upon receptor density and coupling efficiency (McDonald et al., 2003a). As

these parameters are tissue/model dependent, intrinsic activity in different tissues can vary.

Using the ecdysone-inducible expression system containing the human NOP receptor

(hNOP) expressed in Chinese hamster ovary cells they performed [35S]GTPγS binding and

inhibition of adenylyl cyclase studies to examine the activity of a range of partial agonists.

They found that profile of [F/G]N/OFQ(1–13)-NH2 can be manipulated to encompass full

and partial agonism, along with antagonism (McDonald et al., 2003a).

Further modifications of the N/OFQ N-terminus led to the design of

[Nphe1]N/OFQ(1–13)-NH2 by transposition of the Phe1 side chain from the α-carbon of

Phe1 to the N-terminal nitrogen (Guerrini et al., 2000a). This peptide was the first pure

NOP peptide antagonist; it had low potency (pA2 values 6.0–6.4) (Calo et al., 2000a) but

was devoid of any residual agonist activity. This modified N/OFQ peptide selectively

antagonized the effects of N/OFQ in vitro in various isolated tissues and in CHO cells

expressing the human recombinant NOP receptor (Berger et al., 2000b; Calo et al., 2000a;

Hashimoto et al., 2000). In vivo, i.c.v. administration of this peptide inhibited the

pronociceptive and antiopioid actions of N/OFQ (Rizzi et al., 2000) and reversed the

46

effects of N/OFQ on memory impairment (Redrobe et al., 2000), food intake (Polidori et

al., 2000a), and locomotor activity (Rizzi et al., 2001a). This compound, per se, is able to

induce changes opposite to that evoked by N/OFQ such as antinociception (Calo et al.,

2000a), prevention of ibotenate induced neurotoxicity (Laudenbach et al., 2001) and

inhibition of food intake (Polidori et al., 2000b), facilitation of the flexor reflex with no

depression (Xu et al., 2002). Importantly, a study by Di Giannuario and colleagues has

shown that no tolerance develops to the antinociceptive action of this antagonist, unlike

with opioid analgesics, suggesting that NOP antagonists can be developed as a novel class

of supraspinal analgesics (Di Giannuario et al., 2001).

Modification of N/OFQ and N/OFQ(1–13)-NH2 has also produced peptide agonists

more potent than N/OFQ. Okada et al. (2000) reported the synthesis of

[Arg14Lys15]N/OFQ, which had 3-fold greater binding affinity than N/OFQ at human NOP

and was 17 times more potent in the [35S]GTPγS functional assay. [Arg14Lys15]N/OFQ was

the first NOP receptor agonist more potent than the natural ligand, in addition its effects

are long lasting in vivo (Rizzi et al., 2002c).

Guerrini et al. (2001) focused their attention on the Phe4 residue and found that

para-substituted electron-withdrawing groups such as pF and pNO2 increased binding

affinity to NOP receptor 5- and 3-fold, respectively. These agonist peptides were more

potent than N/OFQ at recombinant hNOP and at native NOP receptor sites expressed in

isolated tissues (Bigoni et al., 2002; McDonald et al., 2002). These agonists also display

longer duration of action in vivo in several assays, compared to N/OFQ (Rizzi et al.,

2002b).

Another cyclic peptide having an intramolecular disulphide bridge: cyclo [Cys10,

Cys14]N/OFQ(1-14)-NH2, was reported to be the first conformationally restricted

derivative among the N/OFQ analogues. This peptide has agonist activity and may serve as

a good template for studying the bioactive conformation of N/OFQ (Ambo et al., 2001).

Combination of the cyclic analog with Nphe1 which previously led to pure antagonism,

provided a partial agonist c[Nphe1Cys10,14]N/OFQ(1-14)-NH2 (Kitayama et al., 2003).

Furthermore in a series of structure-activity studies (Guerrini et al., 2005) the

chemical modifications which reduce ([Phe1Ψ(CH2-NH)Gly2]) or eliminate ([Nphe1])

agonist efficacy, in the N/OFQ-NH2 structure, were combined with those which increase

agonist potency i.e. [(pF)Phe4] and [Arg14Lys15]. This study led to the identification of a

very potent antagonist, [Nphe1Arg14Lys15]N/OFQ-NH2 (UFP-101) (Calo et al., 2002b),

and [(pF)Phe4Arg14Lys15]N/OFQ-NH2 (UFP-102) (Carra et al., 2005b), a highly potent and

47

selective full agonist at NOP receptors. The gain in potency was accompanied by slow

onset and relatively long duration of action that was observed in in vitro and especially in

in vivo assays (Carra et al., 2005b; Economidou et al., 2006b). A very potent partial

agonist [Phe1ψ(CH2-NH)-Gly2(pF)Phe4Arg14Arg15]N/OFQ-NH2 (Guerrini et al., 2005) was

also identified.

Regarding the potent and selective antagonist UFP-101 a detailed summary of its

pharmacological characterization was reported by our group (Calo et al., 2005).

Collectively data obtained in vitro in a variety of preparations with different approaches

demonstrated that UFP-101 behaves as a potent, competitive and selective antagonist at

NOP receptors. In vivo, UFP-101 has been tested against N/OFQ in a series of experiments

aimed at the investigation of the role of the N/OFQ-NOP receptor system in regulating

various biological functions including pain transmission, locomotor activity, mood-related

behaviours, drug abuse, food intake and cardiovascular, renal and gastrointestinal function.

It has been demonstrated that UFP-101 antagonizes the following actions of N/OFQ:

hyperalgesia, reversal of stress-induced analgesia, inhibition of locomotor activity,

stimulation of diuresis in mice, bradycardia, hypotension and reduction of plasma

norepinephrine levels in guinea pigs, stimulation of food intake and spinal analgesia in

rats. Moreover UFP-101 (like other selective NOP antagonists) produced antidepressant-

like effects in normal mice in the forced swimming or the tail suspension test. In mice

lacking the NOP receptor gene these actions are absent (Calo et al., 2005).

Recently (Economidou et al. (2006b) verified the hyperphagic effect of 3 new

peptide agonists, termed OS-500, OS-462 and OS-461 which display an affinity for NOP

receptors in the subnanomolar range: no selectivity data were provided.

Previous structure–activity and NMR studies on N/OFQ demonstrated that Aib

substitution of Ala7 and/or Ala11 increases peptide potency through an alpha helix structure

induction mechanism (Zhang et al., 2002). Based on these findings Arduin et al. (2007)

synthesised a series of N/OFQ-NH2 analogues substituted in position 7 and 11 with Ca,a-

disubstituted cyclic, linear and branched amino acids. None of the 20 novel N/OFQ

analogues produced better results than [Aib7]N/OFQ-NH2. Thus, this substitution was

combined with other chemical modifications known to modulate peptide potency and/or

efficacy generating [Nphe1Aib7Arg14Lys15]N/OFQ-NH2 (coded as UFP-111),

[(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-112) and compound [Phe1Ψ(CH2–

NH)Gly2(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-113). These novel peptides behaved

as highly potent NOP receptor ligands showing full (UFP-112) and partial (UFP-113)

48

agonist and pure antagonist (UFP-111) activities in a series of in vitro functional assays

performed on pharmacological preparations expressing native as well as recombinant NOP

receptors (Arduin et al., 2007). In vitro data obtained in the electrically stimulated mouse

vas deferens demonstrated that UFP-112 behaved as a high potency (pEC50 9.43) full

agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-

101 but not naloxone and no longer evident in tissues taken from NOP(-/-) mice. In vivo, in

the mouse tail withdrawal assay, UFP-112 (1–100 pmol, i.c.v.) mimicked the actions of

N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive

effects when given i.t. In both cases, UFP-112 was approximately 100 fold more potent

than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the

hyperphagic effect of N/OFQ producing a bell shaped dose response curve, this effect was

absent in NOP(-/-) mice. Equi-effective high doses of UFP-112 (0.1 nmol) and N/OFQ (10

nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16 h.

N/OFQ produced a clear inhibitory effect which lasted for 60 min while UFP-112 elicited

longer lasting effects (> 6h). UFP-112 (0.1 and 10 nmol/kg, i.v.) produced a marked and

sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a

profound increase in urine flow (Rizzi et al., 2007b).

Small peptide ligands

The small peptides in this group are identified by screening of synthetic peptide

combinatorial libraries. Peptide III-BTD was identified from a combinatorial library of β-

turn constrained peptides (Becker et al., 1999). This conformationally restricted peptide is

a mixed NOP antagonist / opioid agonist (Bigoni et al., 2000b). Five hexapeptides (Ac-

RYYRIK-NH2, Ac-RYYRWK-NH2, Ac-RYYRWR-NH2, Ac-RYYLWR-NH2, Ac-

RYYKWK-NH2) with high affinity and selectivity for the NOP receptor were identified

from a peptide library containing about 52 million compounds made considering all the

natural amino acids except cysteine (Dooley et al., 1997). Similar to [F/G]N/OFQ(1-13)-

NH2, these peptides were partial agonists whose final effects vary from full agonist to

antagonist depending on the tissue and system used in the study (Berger et al., 1999;

Berger et al., 2000a; Dooley et al., 1997; Ho et al., 2000; Mason et al., 2001; Rizzi et al.,

1999a). These hexapeptides, the shortest peptide sequences interacting with the NOP

receptor, have been used as chemical templates for SAR studies. The head to tail

cyclization of Ac-RYYRWK-NH2 produced a drastic decrease in binding affinity

(Thomsen et al., 2000b) while the N-terminal acylation with a pentanoyl group (Judd et al.,

49

2003) or the replacement of the Tyr2,3 residues with (pF)Phe (Judd et al., 2004) led to the

discovery of high affinity low efficacy NOP receptor ligands. The N-terminal alkylation of

the central core YYRW with groups bearing a guanidine function generated a NOP

receptor agonist (Ishiama et al., 2001). Modifications on the Trp (W) were also performed

(Carra et al., 2005a) by our group. Finally, substitution of the C-terminal amide with an

alcoholic function produced Ac-RYYRIK-ol, a NOP receptor ligand that displays high

affinity (pKi 7.91) for NOP receptor expressed in rat brain membranes. Ac-RYYRIK-ol

antagonized N/OFQ effects in the vas deferens while it mimicked N/OFQ action in the

colon. In vivo, the peptide consistently behaved as a NOP receptor agonist mimicking the

supraspinal pronociceptive, orexigenic, and motor inhibiting actions and the spinal

antinociceptive effects of N/OFQ (Gunduz et al., 2006).

SinVax Inc. proposed the compound pentanoyl-RYYRWR-NH2 as NOP receptor

antagonist. In [35S]GTPγS assays performed in CHO cells transfected with human NOP it

displayed a very high affinity with a very low agonist activity (pA2 value of 8.99). When

tested in vivo, this compound had a modest analgesic effect, somewhat less than has been

reported with other NOP antagonists. Moreover this compound inhibited morphine-

induced analgesia suggesting some agonist activity in vivo (Judd et al., 2003).

In order to improve the stability and therapeutic utility of these small peptide

ligands, a novel technology called structure inducing probes (SIP) (Larsen, 1999) was

applied to the hexapeptide Ac-RYYRWK-NH2 (Dooley et al., 1997), resulting in the

design of the peptide Ac-RYYRWKKKKKKK-NH2 (ZP120) (Larsen et al., 2001). ZP120

behaved as potent and selective NOP receptor partial agonist whose in vivo effects are long

lasting (Rizzi et al., 2002a) and, after i.v. administration, confined to periphery. This

pharmacological profile makes ZP120 an interesting drug candidate especially for those

indications (i.e. aquaresis (Kapusta, 2000)) for which NOP partial agonists that produce

renal but not cardiovascular effects are more selective than full agonists which are known

to elicit both renal and cardiovascular actions (Kapusta et al., 2002).

This hypothesis was recently confirmed by Kapusta and collegues (Kapusta et al.,

2005b). The further pharmacological characterization of ZP120 is one of the goals of this

project.

Although the design and pharmacological characterization of peptide ligands for

NOP has facilitated great advances in elucidating the functional role of the N/OFQ-NOP

receptor system, the therapeutic utility of NOP ligands, particularly for neurological

50

disorders, can only be realized with potent non-peptide ligands. This is because as

compared to peptide ligands, non-peptide would be expected to be more resistant to

enzymatic breakdown following oral or parenteral administration and allow greater

penetration into the CNS where they may have therapeutic utility. Many pharmaceutical

companies and different groups have discovered potent non-peptide agonists and

antagonists. These are summarized below.

Non-peptide ligands

Non-peptide ligands are generally discovered via high-throughput screening (HTS)

in pharmaceutical industry laboratories. Since the NOP receptor represents the non-opioid

branch of the opioid family, the search for non-peptide ligands was initiated by examining

small-molecule opiate ligands. Among these compounds are: i) σ-receptor ligands

carbetapentane and rimcazole (Kobayashi et al., 1997); ii) MOP receptor ligands lofentanil

(an anilidopiperidine) and etorphine (an oripavine derivative) (Butour et al., 1997); iii)

anilidopiperidines, morphinans and benzomorphan classes of opiate ligands (Hawkinson et

al., 2000); iv) MOP receptor ligand buprenorphine (Wnendt et al., 1999); v)

naloxonebenzoylhydrazone (NalBzOH) (Noda et al., 1998); vi) the 5-HT partial agonist

spiroxatrine (Zaveri et al., 2001).

The non-peptide NOP receptor ligands can be broadly divided into six structural

classes. It is noteworthy that many of these ligands were first reported in the patent

literature (patents from Pfizer, Banyu Pharmaceutical Co., Hoffmann La Roche,

EuroCeltique S.A., NovoNordisk, Schering-Plough, Smith Kline Beecham, Japan Tobacco

Inc, Toray Industries, etc). However, the biological data of some of them are still not

available, thus making it difficult to define clearly the structural requirements for NOP

receptor affinity and selectivity.

i) Morphinan-based ligands: Among this group TRK-820 was reported to antagonize

the effects of N/OFQ on cAMP accumulation in CHOhNOP cells (Seki et al., 1999). Thus,

the morphinan skeleton may provide a good lead for a unique profile of NOP antagonism

coupled with opioid agonist activity for a novel class of analgesics.

ii) Benzimidazopiperidines: The first non-peptide pure NOP antagonist to be reported

was a benzimidazolinone, J-113397 (Figure 8), reported by Kawamoto et al. (1999) and

patented by Banyu Pharmaceutical Co. (Ozaki et al., 1998). J-113397 was shown to bind

51

with nanomolar affinity to NOP receptors and to display 100-300 fold selectivity over

classical opioid receptors (Hashiba et al., 2001; Ozaki et al., 2000). J-113397 antagonized

N/OFQ effects at human NOP receptor in a competitive manner with pA2 values in the

range of 7.5 – 8.9 in cAMP and [35S]GTPγS assays (Bigoni et al., 2000a; Hashiba et al.,

2002a; Hashiba et al., 2002b; Ozaki et al., 2000). The selective antagonist properties of J-

113397 were confirmed at native NOP receptors expressed in isolated tissues (Bigoni et

al., 2000a; Tada et al., 2002) and in brain preparations evaluated with biochemical

(Olianas et al., 2002), neurochemical (Marti et al., 2003; Rominger et al., 2002) and

electrophysiological (Chiou et al., 2002; Luo et al., 2002) techniques. J-113397 was also

investigated in vivo where, in the range of 1-30 mg/kg, it prevented the actions of N/OFQ

on pain transmission (Ko et al., 2002a; Ozaki et al., 2000; Ueda et al., 2000), on airways

(Corboz et al., 2001) and the cough reflex (Bolser et al., 2001; McLeod et al., 2002), and

on gastrointestinal functions (Ishihara et al., 2002; Tada et al., 2002). Moreover J-113397

produced per se pronociceptive effects in the rat (Yamamoto et al., 2001) and mouse

(Rizzi et al., 2006) formalin test, antidepressant like effects (similar to NOP receptor

peptide antagonists (Gavioli et al., 2003; Redrobe et al., 2002)) in the forced swimming

test (Redrobe et al., 2002), reduction of kainate induced seizures (Bregola et al., 2002),

potentiation of buprenorphine analgesia in wild type but not in NOP knockout mice (Lutfy

et al., 2003), and facilitation of striatal dopamine release and locomotor performance on

the rotarod in rats (Marti et al., 2004b). This latter effect was later confirmed in 6-

hydroxydopamine lesioned animals (Marti et al., 2005).

To date, J-113397 represents the most potent and selective non-peptide NOP

receptor antagonist widely used in pharmacological studies. However, the synthesis,

purification, and enantiomer separation of this molecule, which contains two chiral centers,

is rather difficult and low-yielding. A series of simplified J-113397 analogues was

synthesized and tested to investigate the importance of the stereochemistry and the

influence of the substituents at position 3 of the piperidine nucleus and on the nitrogen

atom of the benzimidazolidinone nucleus. The compound coded as Trap-101 (Figure 8), an

achiral analogue of J-113397, combines a pharmacological profile similar to that of the

parent compound with a practical, high-yielding preparation (Trapella et al., 2006). In in

vitro N/OFQ sensitive preparations Trap-101 was a NOP selective antagonist with a

potency 2-3 fold lower than the reference compound J-113397. In vivo, Trap-101 changed

motor activity in naive rats and mice and alleviated parkinsonism in 6-hydroxydopamine

hemilesioned rats: Trap-101 stimulated motor activity at 10 mg/kg and inhibited it at 30

52

mg/kg (Marti et al., 2008). Trap-101 could be use as a novel template for a structure-

activity study aimed at establishing the importance of both the C3 hydroxymethyl function

and of the benzimidazolidinone nitrogen substituent.

J-113397 Trap-101

Figure 8. Structures of non-peptide NOP antagonist J-113397 and Trap-101.

Pfizer has also reported a new series of benzimidazoles as NOP agonists, among

them PCPB and MCOPPB (Figure 9). PCPB bound to the NOP receptor in mouse brain

membranes (Ki = 0.12 nM) and to recombinant human NOP receptor (Ki = 2.1 nM). Orally

administered PCPB (30 mg/kg) exhibited anxiolytic activity in mice subjected to the Vogel

conflict test that was comparable to the maximal response induced by diazepam (Hirao et

al., 2008a). MCOPPB showed a high affinity for the human NOP receptor (pKi = 10.07)

and selectivity for the NOP receptor over other members of the opioid receptor family: 12,

270 and >1000 fold more selective for the NOP receptor than for the MOP, KOP, and DOP

receptors, respectively; in vivo MCOPPB (10 mg/kg, p.o.) elicited anxiolytic-like effects in

mice without affecting locomotor activity or memory. On the other hand, the

benzodiazepine-type anxiolytic agent diazepam caused memory deficits (Hirao et al.,

2008b).

53

PCPB MCOPPB

Figure 9. Structures of non-peptide NOP agonist PCPB and MCOPPB.

iii) Spiropiperidines: Hoffmann La Roche disclosed a series of 1,3,8-

triazaspiro[4,5]decan-4-ones, discovered through high throughput screening. Among them

Ro 65-6570 and Ro 64-6198 were two of those ligands widely used as pharmacological

tools (Adam et al., 1998) (Figure 10). Although Ro 65-6570 was found to show anxiolytic

effects (Wichmann et al., 1999), it was only 5 to 10-fold selective over opioid receptors

(Hashiba et al., 2001). Ro 64-6198, on the other hand, is far more selective and has shown

an impressive anxiolytic profile comparable to benzodiazepines, in several in vivo anxiety

paradigms (Jenck et al., 2000; Le Pen et al., 2002). As an agonist only slightly less potent

than N/OFQ itself (Hashiba et al., 2002b), Ro 64-6198 can potentially be used as a

therapeutic agent in disorders where a NOP agonist may prove beneficial, such as anorexia

(Ciccocioppo et al., 2002), anxiety (Jenck et al., 2000), and inhibition of drug reward

pathways (Dautzenberg et al., 2001). However, Ro 64-6198 was found not to affect

cocaine-induced conditioned place preference (Kotlinska et al., 2003). Moreover, at higher

doses, Ro 64-6198 was found to have affinity for dopamine and sigma receptors (Jenck et

al., 2000) and increased alcohol drinking in genetically selected alcohol-preferring

Marchigian Sardinian rats while other NOP agonists such as UFP-102 and UFP-112 reduce

alcohol drinking; an effect probably induced by residual agonist activity of this compound

at MOP receptors (Economidou et al., 2006a). For review see Shoblock (2007).

54

N

HN

N

O

N

HN

N

O

H

Ro 65-6570 Ro 64-6198

Figure 10. Structures of Hofmann-La Roche lead compounds Ro 65-6570 and Ro 64-6198.

Interestingly, Novo Nordisk has also reported on the synthesis and characterization

of 1,3,8-triazaspirodecanones, similar to the Roche compounds, starting with spiroxatrine

as their lead (Thomsen et al., 2000a). Their best ligand, NNC 63-0532 had binding affinity

of 6.3 nM against human NOP but only a 12-fold selectivity for NOP over classical opioid

receptors. This low selectivity was also seen in electrically stimulated mouse vas deferens

where NNC 63-0532 produced a concentration-dependent inhibition of the electrically

induced twitches showing, in comparison with N/OFQ, lower potency and higher maximal

effects. In addition, contrary to N/OFQ, the effects of NNC 63-0532 were insensitive to the

NOP selective antagonist UFP-101 but were prevented by naloxone (Guerrini et al., 2004).

Recently it was seen that NNC 63-0532 (0.01 nM-10 µM) like N/OFQ induces a

concentration-dependent endocytosis and recycling of the N/OFQ receptor. This

mechanism contributes to maintain receptor signaling as it counteracts desensitization

development and enhances a compensatory upregulation of adenylyl cyclase activity

(Spampinato et al., 2006).

iv) Aryl piperidines: Designing compounds in this group led several pharmaceutical

companies (Schering Plough, Roche etc) to obtain patents. Recently SB-612111 was

patented by GlaxoSmithKline (Zaratin et al., 2004) (Figure 11). The results describe SB-

612111 as a high affinity and broadly selective NOP receptor antagonist in vitro and in

vivo. Furthermore SB-612111 can resensitize mice to morphine in animals which had been

chronically treated with opiate, suggesting utility of this class of NOP receptor antagonist

in prolonging the analgesic action of morphine (Zaratin et al., 2004). SB-612111 was

synthesized by our laboratories and investigated in vitro and in vivo. In vitro SB-612111

55

displayed subnanomolar affinity for the NOP receptor and high selectivity over classical

opioid receptors (Spagnolo et al., 2007; Zaratin et al., 2004). Functional studies

([35S]GTPγS binding and cAMP accumulation) in CHO cells expressing the human NOP

receptor demonstrated pure, competitive and high potency antagonism exerted by this

molecule against N/OFQ (pKB value of 9.70 and 8.63 in the [35S]GTPγS binding and

cAMP accumulation experiments, respectively (Spagnolo et al., 2007). In isolated

peripheral tissues of mice, rats, and guinea pigs and in mouse cerebral cortex

synaptosomes preloaded with [3H]5-HT, SB-612111 competitively antagonized the

inhibitory effects of N/OFQ, with pA2 values in the range of 8.20 to 8.50 (Spagnolo et al.,

2007). In vivo, in the mouse tail withdrawal assay, SB-612111 given i.p. up to 3 mg/kg

prevented the pronociceptive and the antinociceptive action of 1 nmol of N/OFQ given

i.c.v. and i.t., respectively (Rizzi et al., 2007a). In food intake studies performed in sated

mice, SB-612111 (1 mg/kg i.p.) had no effect on food consumption but fully prevented the

orexigenic effect of 1 nmol of N/OFQ i.c.v. (Rizzi et al., 2007a). In the mouse forced

swimming and tail suspension tests, SB-612111 (1-10 mg/kg) reduced immobility time.

The antidepressant-like effect elicited by SB-612111 in the forced swimming test was

reversed by the i.c.v. injection of 1 nmol of N/OFQ and was no longer evident in mice

knockout for the NOP receptor gene (Rizzi et al., 2007a). In conclusion, SB-612111 is

among the most potent and NOP-selective non-peptide antagonists identified to date.

OH

N

Cl

Cl

SB-612111

Figure 11. Structure of SB-612111.

v) 4-Aminoquinolines: These are an entirely novel chemical class of NOP ligands

disclosed by Japan Tobacco Inc. in a patent (Shinkai et al., 2000). The optimized ligand,

JTC-801 (Figure 12), was obtained through an extensive structure-activity study of lead

compound 25. Detailed pharmacological studies with JTC-801 were recently reported

(Yamada et al., 2002). Its binding affinity for hNOP was 44.5 nM. It completely

56

antagonized the inhibition of cAMP accumulation by N/OFQ. Furthermore, when

administered in vivo orally or i.v., at doses of 0.1-1 mg/kg, it antagonized N/OFQ

induced allodynia in mice and increased latency in the mouse hot plate test. These

effects were not inhibited by naloxone.

O

HN

O

N

H2N JTC-801

Figure 12. Structure of JTC-801, a novel Japan Tobacco Inc compound.

JTC-801 was chosen as a candidate for clinical trials for analgesia because of its

oral bioavailability profile, which was more favourable than that of some other more potent

analogs in this series. It was seen that JTC-801 alleviates heat-evoked hyperalgesia in

chronic constriction injury rats (Suyama et al., 2003).

vi) N-benzyl-D-proline: Recently Banyu Pharmaceuticals discovered a novel class of

NOP antagonists using a focused library approach starting from a moderately active hit

compound found in their chemical collection. The N-benzyl-D-proline analogue

(Compound 24) (Figure 13) showed significantly improved antagonistic activity when

compared with other reported NOP antagonists and showed good brain penetrability and in

vivo antagonistic activity (Goto et al., 2006).

NNH

N

O

O Compound 24

Figure 13. Structure of Compound 24, a novel Banyu Pharmaceuticals compound.

57

Based on this novel antagonist structure we performed a structure activity analysis

of Compound 24, focusing on its N-benzyl-D-proline, amide bond and benzoisofurane

moieties; this latter structure was substituted with moieties taken from known non-peptide

NOP ligands such as Ro 64-6198, SB-612111 and J-113397. Twelve new derivatives were

synthesized and evaluated for their ability to bind the human recombinant NOP receptor.

The molecule showing the highest affinity (Compound 35) has been further characterized.

The pharmacological characterization of both Compound 24 and Compound 35 is one of

the major goals of this thesis.

Finally, there is a recent description of a 4-aryl-tropane NOP agonist, SCH 221510

(Varty et al., 2008) a new molecule discovered by Schering-Plough (Figure 14). SCH

221510 binds with high affinity (Ki 0.3 nM) to the NOP receptor and was shown to be

anxiolytic with a reduced side-effect profile when compared with benzodiazepines (Varty

et al., 2008).

N

OH

SCH 221510

Figure 14. Structure of SCH 221510.

58

Table 2. NOP receptor ligands available to date.

N/OFQ-RELATED PEPTIDE ANALOGUES NON-

PEPTIDES

AG

ON

ISTS

♦ N/OFQ

♦ N/OFQ-NH2

♦ [Tyr1]N/OFQ

♦ N/OFQ(1-13)-NH2

♦ [Arg14Lys15]N/OFQ

♦ [(pF)Phe4]N/OFQ(1-13)-NH2

♦ [(pF)Phe4Arg14Lys15]N/OFQ-NH2 (UFP-102)

♦ [(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-112)

♦ OS-461, OS-462, OS-500

♦ Ro 65-6570

♦ NNC-63-0532

♦ Ro 64-6198

♦ SCH 221510

♦ PCPB

♦ MCOPPB

PAR

TIA

L A

GO

NIS

TS

♦ [Phe1ψ(CH2-NH)-Gly2]N/OFQ(1-13)-NH2

♦ [Phe1ψ(CH2NH)Gly2(pF)Phe4Arg14Lys15]N/OFQ-NH2 (UFP-103)

♦ [Phe1ψ(CH2NH)Gly2(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-113)

AN

TAG

ON

ISTS

♦ [Nphe1]N/OFQ(1-13)-NH2

♦ [Nphe1Arg14Lys15]N/OFQ-NH2 (UFP-101)

♦ [Nphe1(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-111)

♦ NalBzoH

♦ J-113397

♦ JTC-801

♦ SB-612111

♦ Trap-101

♦ Compound 24

59

1.9 Aims

The main objectives of this work have been to:

♦ Produce a detailed characterization of the pharmacological profile of NOP receptors

coupled with calcium signalling via the chimeric protein Gαqi5 using a large panel of

full and partial agonists as well as pure antagonists. [Ca2+]i levels were monitored using

the fluorometer FlexStation II. This has the potential to provide a rapid screening tool

for novel NOP ligands.

♦ Further investigate the pharmacological profile of ZP120 using mouse and rat

preparations, J-113397 and UFP-101, as well as NOP(-/-) mice.

♦ Fully characterize the pharmacological profile of the novel non-peptide NOP

antagonist Compound 24.

♦ Screen a new series of non-peptide ligands deriving from SAR studies on the potent

antagonist Compound 24. Compound 35, the best compound of this series, has been

further investigated.

To pharmacologically characterize these molecules, in vitro studies on N/OFQ-

sensitive isolated tissues from different species and on transfected cells expressing the

recombinant NOP and classical opioid receptors, were performed. In addition, in vitro

experiments were also assessed in CHO cells co-expressing the recombinant NOP and

classical opioid receptors and the chimeric protein Gαqi5. Moreover, some compounds

were studied using an in vivo assay in mice, the tail withdrawal assay. Some in vitro

and in vivo experiments were performed using NOP(+/+) and NOP(-/-) mice.

60

2. MATERIALS & METHODS

2.1 Drugs and reagents

The peptides used in this study were synthesized in the laboratory of Prof Salvadori

(Department of Pharmaceutical Sciences and Biotechnology Centre, University of Ferrara)

using standard solid-phase synthesis techniques and purified using High Pressure Liquid

Chromatography, according to previously published methods (Guerrini et al., 1997).

Amino acids, protected amino acids, and chemicals were purchased from Bachem,

Novabiochem, Fluka (Switzerland) or Chem-Impex International (U.S.A).

The compound [F/G]N/OFQ(1-13)-NH2 was prepared as described by Guerrini et

al. (2003), [Nphe1]N/OFQ(1-13)-NH2 was obtained by the shift of the side chain of Phe1

from the C to the N atom in the template N/OFQ(1-13)-NH2. NNC 63-0532-COOH was

obtained following literature methods (Watson et al., 1999) and UFP-111, UFP-112 and

UFP-113 were prepared as previously described (Arduin et al., 2007). Ac-RYYRIK-NH2,

Ac-RYYRIK-ol and all the other modified hexapeptides were synthesized accordingly to

Kocsis et al. (2004) in the laboratory of Dr Anna Magyar (Research Group of Peptide

Chemistry, Eötvös Loránd University, Budapest, Hungary) using solid-phase peptide

synthesis techniques.

ZP120 was provided by Zealand Pharma (Copenhagen, Denmark), [D-Pen2,D-

Pen5]enkephalin (DPDPE) and dynorphin A were purchased from NeoMPS (Strasbourg,

France).

Ro 64-6198 was provided by Dr. Wichmann (Hoffmann-La Roche, Basel,

Switzerland). The non-peptide compound Compound 24 was synthesized following the

procedures described in detail by Goto et al. (2006).

J-113397 was prepared as a racemic mixture, according to De Risi et al. (2001),

Trap-101 is obtained through treatment with LiAlH4 on a common intermediate from the J-

113397 synthesis. The compound SB-612111 was synthesized following the experimental

conditions and protocols described in detail in the patent (WO 03/040099 A1) by Palombi

et al. (2003).

Captopril, amastatin, bestatin, phosphoramidon, naloxone, bovine serum albumin

(BSA), guanosine 5’-O-(3-thiotri-phosphate) (GTPγS), GDP, unlabelled GTPγS, bacitracin

and probenecid were from Sigma Chemical Co. (Poole, U.K.) or E. Merck (Darmstadt,

Germany). All tissue culture media and supplements were from Invitrogen (Paisley, U.K.).

[35S]GTPγS (1250 Ci mmol-1), [3H]Diprenorphine ([3H]DPN, 75-133 Ci/mmol) were from

61

Perkin Elmer Life Sciences (Boston, Mass., USA) and [leucyl-3H]N/OFQ ([3H]-N/OFQ,

150 Ci/mmol) was from Amersham Pharmacia Biotech (Buckinghamshire, UK). All other

consumables and reagents were of the highest purity available.

For in vitro experiments, the peptides were solubilized in H2O and stock solutions

(1 mM or 2 mM) were stored at -20 °C until use; the non-peptide compounds were

solubilized in dimethyl sulfoxide at a final concentration of 10 mM, and the successive

dilutions were made in saline or water, stock solutions were kept at -20 °C until use. For in

vivo studies, Compound 24 was dissolved in 2% DMSO and 10% encapsin

(hydroxypropyl-beta-cyclodextrin) just before performing the experiment.

2.2 Buffer composition

• Tissue culture buffers:

Harvest: HEPES (10 mM), EDTA (1.1 mM), NaCl (154 mM), pH 7.4 with NaOH.

• [35S]GTPγS buffers:

[35S]GTPγS Homogenising: Tris-HCl (50 mM), EGTA (0.2 mM), pH 7.4 with

NaOH.

[35S]GTPγS Assay: Tris-HCl (50 mM), EGTA (0.2 mM), NaCl (100 mM), MgCl2 (1

mM), pH 7.4 with NaOH.

• [3H]-N/OFQ competition buffers:

Wash/Homogenising: Tris-HCl (50 mM), MgSO4 (5 mM), pH 7.4 with KOH.

Assay: Tris-HCl (50 mM), MgSO4 (5 mM), pH 7.4 with KOH, 0.5% BSA.

• [3H]-Diprenorphine competition buffers:

Wash/Homogenising: Tris-HCl (50 mM), pH 7.4 with KOH.

Assay: Tris-HCl (50 mM), pH 7.4 with KOH, 0.5% BSA.

• Calcium mobilization assay buffers:

HBSS (Hanks’ Balanced Salt Solution): KCl (5.4 mM), KH2PO4 (0.44 mM), NaCl

(137 mM), NaHCO3 (4.2 mM), Na2HPO4 x 7 H2O (0.25 mM), CaCl2 (1.3 mM),

MgSO4 x 7 H2O (1 mM), glucose (5 mM).

62

Loading solution: cell culture medium, HEPES (20 mM), probenecid (2.5 mM),

calcium-sensitive fluorescent dye Fluo 4AM (3 µM), pluronic acid (0.01%).

Dye loading solution: HBSS, HEPES (20 mM), probenecid (2.5 mM), Brilliant Black

(500 µM).

• Bioassay buffers composition:

The tissues were suspended in the following Krebs solution (mM): NaCl 118.5, KCl

4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2.5, glucose 10. For the experiments

on the mouse vas deferens and rat vas deferens, a Mg++-free and 1.8 mM CaCl2 Krebs

solution were used, respectively. For the experiments on guinea pig ileum the normal

medium was added with hexamethonium (0.0008%) and benadryl (0.00001%).

2.3 In vitro studies

CHO expressing the recombinant NOP/DOP/MOP/KOP receptors

2.3.1 Cell harvesting and membranes preparation

Chinese Hamster Ovary cells (CHO) stably expressing the human NOP receptor

(CHOhNOP) were supplied by Dr. Maithe Corbani (Institut de Pharmacologie et de Biologie

Structurale, Toulouse, France). Cells were cultured in media consisting of Dulbecco's

MEM/HAMS F12 (50/50) supplemented with 5% foetal calf serum (FCS), penicillin (100

IU/ml), streptomycin (100 μg/ml), fungizone (2.5 μg/ml), geneticin (G418; 200 μg/ml) and

hygromycin B (200 μg/ml) at 37˚C in 5% CO2/humidified air. CHO cell stocks expressing

the human DOP/MOP/KOP receptors (CHOMOP/DOP/KOP) were kindly provided by Dr.

Larry Toll (SRI International, Menlo Park, CA, USA) and maintained in Ham F12

containing 10% FCS, 100 IU/ml P, 100 μg/ml S and 400 μg/ml G418, for CHO non-

transfected cells G-418 and hygromycin B were omitted. Cell cultures were kept at 37˚C in

5% CO2/humidified air. In all cases experimental cultures were free from selection agents

(hygromycin B, G418). When confluence was reached (3-4 days), cells were sub-cultured

as required using trypsin//EDTA and used for experimentation. Cells were harvested from

sterile tissue culture flasks using harvest buffer and gentle agitation. Cells were suspended

in either wash buffer (displacement assay) or homogenising buffer (GTPγ[35S] assay),

homogenised using an Ultra Turrax, for 10 seconds followed by 6 consecutive 1-second

63

bursts. The homogenate was then centrifuged at 13,500 rpm for 10 min at 4°C, this was

carried out a total of three times. The membrane fraction was resuspended in an

appropriate volume of assay buffer and the total protein content determined as set out

below.

2.3.2 Displacement binding assay

5 μg protein of CHOhNOP homogenate were assayed in a total volume of 0.5 ml

comprising competition homogenising buffer supplemented with 0.5% (w:v) BSA, 10 μM

peptidase inhibitors (amastatin, bestatin, captopril and phosphoramidon), 0.2 nM [Leucyl-3H]N/OFQ and 100 nM – 0.1 pM of competing ligands. Non-specific binding (NSB) was

determined in the presence of 1 μM N/OFQ. 50 µg (CHOhMOP), 25 µg (CHOhDOP) and 40

µg (CHOhKOP) membrane protein were incubated in 0.5 ml homogenising buffer

supplemented with 0.5% BSA, approximately 0.7 nM [3H]Diprenorphine. NSB binding

was defined in the presence of 10 µM naloxone. Reactions were incubated for 1 hour at

room temperature and harvested under vacuum filtration using a Brandel cell harvester.

Whatman GF/B filters were soaked in 0.5% polyethylenimine, to reduce NSB, and loaded

onto the harvester wet. Radioactivity was determined following filter extraction (8 hours,

Optiphase Safe) using liquid scintillation spectroscopy.

2.3.3 [35S]GTPγS stimulation binding assay

Experimentation was performed essentially as described by Berger et al. (2000b).

Freshly prepared CHOhNOP membranes (20 μg) were incubated in 0.5 ml volumes of buffer

consisting Tris (50 mM), EGTA (0.2 mM), GDP (100 μM), bacitracin (0.15 mM), BSA (1

mg/ml), peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon; 10 μM),

[35S]GTPγS (~150 pM) and ligands in the concentration range of 10-12 – 10-5 M. NSB was

determined in the presence of 10 μM unlabelled GTPγS. Assays were incubated for 1 h at

30oC with gentle shaking and bound and free radiolabel were separated by vacuum

filtration onto Whatman GF/B filters. Polyethylenimine was not used. In all cases

radioactivity was determined following filter extraction (8 hours) using liquid scintillation

spectroscopy.

64

2.3.4 Protein assay

The protein concentration was determined for membrane fractions using the method

of Lowry (Lowry et al., 1951): BSA protein standards at set concentrations of 0, 50, 100,

150, 200, 250 μg protein/ml were made up in 0.1 M NaOH. Samples of unknown protein

concentration were diluted in 0.1 M NaOH. 0.5ml volumes of standards and samples were

incubated for 10 min in 2.5 ml of solution consisting of, A (NaHCO3 in 0.1 M NaOH) B

(1% CuSO4) and C (2% Na+ K+ tartrate) mixed to the ratio 100:1:1. Folin’s reagent

(diluted 1:4 in dH2O) was then added and incubated at room temperature for a further

30min. The absorbance at 750 nm for standards and samples was then determined using a

spectrophotometer. Linear regression of the known BSA protein concentrations was used

to produce a standard curve (Figure 15) from which sample protein concentrations were

determined.

0 50 100 150 200 250 3000.0

0.2

0.4

0.6

r2 = 0.99

[Protein] (μg/ml)

Abs

orba

nce

(750

nm)

Figure 15. Example of protein assay standard curve used to determine the protein mass of unknown samples.

Calcium mobilization assay

2.3.5 Experimental protocols

CHO cell lines stably co-expressing MOP/DOP/KOP/ NOP receptors and the C-

terminally modified Gαqi5 were generated as described by Camarda et al. (2009).

CHOhMOP, CHOhDOP, CHOhKOP and CHOhNOP stably expressing the Gαqi5 protein were

65

seeded at a density of 40,000 cells/well into 96-well black, clear-bottom plates. After 24

hours incubation the cells were loaded with medium supplemented with 2.5 mM

probenecid, 3 µM of the calcium sensitive fluorescent dye Fluo-4 AM and 0.01% pluronic

acid, for 30 min at 37 °C (Figure 16). Afterwards the loading solution was aspirated and

100 µl/well of assay buffer: HBSS buffer supplemented with 20 mM HEPES, 2.5 mM

probenecid and 500 µM Brilliant Black was added. Stock solutions (1 mM) of ligands were

made in distilled water and stored at -20 °C. Serial dilutions of ligands for experimental

use were made in HBSS/HEPES (20 mM) buffer (containing 0.02% BSA fraction V).

After placing both plates (cell culture and compound plate) into the FlexStation II

(Molecular Device, Union City, CA 94587, US), fluorescence changes were measured at

room temperature. On-line additions were carried out in a volume of 50 µl/well.


Figure 16. Diagram depicting the experimental protocol of the calcium mobilization assay. Cells are incubated with Fluo-4 AM, de-esterification of the ester group (AM) traps the dye in the cells and further leakage of the dye is prevented by blockage of organic anion-transport inhibitors using probenecid. Background fluorescence is reduced by addition of Brilliant Black dye which blocks extracellular signalling from any leaked Fluo-4.

2.3.6 Cell counting

Accurate numbers in a cell suspension can be calculated by counting the cells in a

cell counting chamber (Burker’s chamber, Figure 17). A small volume of the cell

suspension (10 µl) was pipetted onto the chamber, the capillary action under the cover slip

66

will draw the suspension into the counting chamber. The space between the cover slip and

the counting chamber ensures a specific volume of cell suspension is present.


Figure 17. Burker’s chamber.

Under a microscope the number of cells in diagonally opposite counting areas were

counted, Figure 18.


Figure 18. Schematic representation of the counting grid of the Burker’s chamber.

The Burker’s chamber is formed of 3x3 major squares, each of these major squares

is subdivided into a grid of 4x4 squares. The number of cells present in three major cells

are counted, cells in contact with two of the squares sides are included and the average

taken. The volume of a major square is 0.1 mm3 which is equal to 0.0001 ml. To determine

the number of cells per ml the average number of cells determined is increased by a factor

of 104.

2.3.7 Instruments

[Ca2+]i levels were monitored using a FlexStation II fluorimeter (Figure 19). The

FlexStation II system includes:

67

• Xenon-lamp light source

• Automatic eight-channel pipettor

• Tip rack drawer

• Compound plate drawer

• Reading chamber drawer

The Xenon-lamp light source and dual monochromators permit the use of

essentially all dual-wavelength dyes for functional cellular assays.


Figure 19. Diagram of FlexStation II used for calcium mobilization assay.

Isolated tissues

The in vitro experiments were performed on mouse vas deferens (mVD), rat vas

deferens (rVD) and guinea pig ileum (gpI). The animals (Morini, Reggioemilia, Italy) were

handled according to guidelines published in the European Communities Council

directives (86/609/EEC), National regulation (D.L 116/92). They were housed in 425 x

266 x 155 mm cages (Techniplast, Milan, Italy), fifteen animals/cage, under standard

conditions (22ºC, 55 % humidity, 12-h light/dark cycle, light on at 7:00 am) with food

(MIL, standard diet; Morini, Reggio Emilia, Italy) and water ad libitum.

68

2.3.8 Tissue preparation

Tissues were taken from male Swiss mice (25-30 g), guinea pigs (300-350 g) and

Sprague Dawley rats (300-350 g). On the day of the experiments the animals were killed

by a lethal injection of urethane. From the mouse and rat the prostatic portion of the vas

deferens was isolated, and prepared according to Hughes et al. (1975) and Schulz et al.

(1979), respectively; from the guinea pig segments of ileum (1.5-2 cm in length) were

taken as described by Paton (1957). The tissues were suspended in 5 ml organ baths

containing heated Krebs solution oxygenated with 95% O2 and 5% CO2 (pH 7.4). The

temperature was set at 33 °C for the mVD and at 37 °C for the other tissues (Hughes et al.,

1975; Paton, 1957; Schulz et al., 1979). A resting tension of 0.3 g was applied to the mVD,

1 g to the gpI and rVD.

2.3.9 Experimental protocols

The mVD, gpI and rVD were continuously stimulated through two platinum ring

electrodes with supramaximal voltage rectangular pulses of 1 msec duration and 0.05 Hz

frequency. The electrically evoked contractions (twitches) were measured isotonically with

a strain gauge transducer (Basile 7006; UgoBasile s.r.l., Varese, Italy). After an

equilibration period of about 60 min the contractions induced by electrical field stimulation

were stable; at this time, cumulative concentration-response curves to N/OFQ, N/OFQ

related peptides, or to opioid ligands were performed (0.5 log unit steps). The

concentration-response curve consists of progressive administration of increasing

concentrations of peptide without changing the Krebs solution in which the tissue is

bathed; the injection of a certain concentration of ligand must be done only when the

previous concentration produced a stable effect (plateau). About 1 hour with 3 changes of

Krebs solution (wash out) is needed for the tissue to recover the original twitch.

When required, in all the preparations described above, the NOP receptor

antagonists, at adequate concentrations, were added to the medium 15 min before

performing the challenge with agonists.

2.3.10 Instruments

For the in vitro bioassays two chamber-glass baths for isolated organs were utilized

(Figure 20). The outer chamber contains water heated at 33 or 37 °C, while the inner

chamber contains 5 ml of oxygenated Krebs solution. One end of the tissue is fixed to the

69

bottom side of the inner chamber and the other end is linked to a force transducer by a

surgery thread. The role of the transducer is to convert the mechanical signal into an

electrical signal that is then amplified and recorded with a PC-based acquisition system

Power Lab 4/25 (model ML845, ADInstrument, USA).


Figure 20. Diagram of the tissue chamber used for isolated tissue assays.

70

2.4 In vivo studies

Experimental protocol

2.4.1 Animals

Male Swiss and male CD1/C57-BL6J/129 NOP(+/+) and NOP(-/-) mice weighing

20-25 g were used. All transgenic animals were genotyped by PCR. Details of the

generation and breeding of mutant mice have been published previously (Gavioli et al.,

2003). The animals were handled as described above in the isolated tissues section.

I.c.v. injections (2 µl/mouse) were given, under light isofluoran anesthesia (just

sufficient to produce a loss of the righting reflex), in the left ventricule according to the

procedure described by Laursen and Belknap (Laursen et al., 1986). Briefly, the syringe

was held at an approximate 45° angle to the skull. Bregma was found by lightly rubbing

the point of the needle over the skull until the suture was felt. Once found, care was taken

to maintain the approximate 45° angle and the needle was inserted about 2 mm lateral to

the midline. The skull is relatively thin at this point, so only mild pressure was required to

insert and remove the needle. The solutions of drugs were then injected slowly (2 µl in

about 20 s).

I.t. injections (5 µl/mouse) were adapted according to the method of Hylden and

Wilcox (Hylden et al., 1980). A 28-gauge stainless steel needle attached to a 50 µl 65

Hamilton microsyringe was inserted, with an angle of about 20° in the spinal subarachnoid

space between the L5 and L6 segments in mice. 1-3 hours before the i.t. injection a caudal

cutaneous incision was performed under isofluoran anesthesia.

I.p. injections 100 µl/mouse of non-peptide compounds were given 30 min before

N/OFQ administration.

2.4.2 Tail withdrawal assay

All experiments were started at 10.00 a.m. and performed according to the

procedure previously described in detail (Calo et al., 1998b). Briefly, the mice were placed

in a holder and the distal half of the tail was immersed in water at 48 °C. Withdrawal

latency time was measured by an experienced observer blind to drug treatment. A cut off

time of 20 s was chosen to avoid tissue damage. For each experiment four mice were

randomly assigned to each experimental group, and the experiment was repeated at least

four times: therefore each experimental point is the mean of the results obtained in >16

mice.

71

Tail-withdrawal latency was determined immediately before and 5, 15, 30, and 60

min after i.c.v. or i.t. injection of saline (control) or N/OFQ (1 nmole). Compound 24 (10

mg/kg) was given i.p. 30 min before N/OFQ administration. Increased and decreased tail

withdrawal latencies compared with baseline indicated antinociceptive and pronociceptive

effects, respectively.

2.5 Data analysis and terminology

All data are expressed as means ± standard error of the mean (s.e.m.) of n

experiments. For potency values 95% confidence limits were indicated. Data have been

statistically analyzed with the Student’s t test for unpaired data or one way ANOVA

followed by the Dunnett’s test, as specified in table and figure legends; p values less than

0.05 were considered to be significant. The pharmacological terminology adopted in this

manuscript is consistent with the IUPHAR recommendations (Neubig et al., 2003).

[35S]GTPγS data are expressed as a stimulation factor i.e. the ratio between agonist-

stimulated [35S]GTPγS specific (minus NSB) binding and basal specific binding. Receptor

binding data are expressed as pKi derived from the Cheng and Prusoff (Cheng et al., 1973)

equation:

Ki = IC50 / (1+([R]/KD))

where IC50 is the concentration of the competitor producing 50 % displacement, [R] is the

concentration of the radiolabel and KD is the radiolabel affinity for the receptor under

investigation. The [3H]N/OFQ KD was 83 pM while those of [3H]Diprenorphine were 125,

323, and 134 pM (in-house laboratory values) at MOP, DOP, and KOP, respectively. pKi is

the antilogarithm of the Ki values obtained after the calculations.

Calcium mobilization data are expressed as fluorescence intensity units (FIU) in

percent over the baseline. Isolated tissue data are expressed as percent of the twitch

induced by electrical field stimulation.

Agonist potencies are given as pEC50 = the negative logarithm to base 10 of the

molar concentration of an agonist that produces 50% of the maximal possible effect of that

agonist. Concentration response curve to agonists were fitted with the following equation:

Effect = baseline + (Emax-baseline)/(1+10^((LogEC50 - X)*HillSlope))

72

where X is the agonist concentration.

The Emax is the maximal effect that an agonist can elicit in a given tissue. In the

electrically stimulated tissues, the Emax of agonists is expressed as % of inhibition of the

control twitch.

Antagonist potencies are expressed in terms of pA2. pA2 is the negative logarithm

to base 10 of the antagonist molar concentration that makes it necessary to double the

agonist concentration to elicit the original response (Schild, 1997). The pA2 values are

calculated using Schild’s linear regression, that correlates the log of concentrations of

antagonists (x axis) to the log of (CR-1) (y axis), where CR is the ratio between the EC50

(nM) values of agonist, in the presence and in absence of antagonist. The value of x for

y=0 represents the pA2 value, and the slope not significantly different from the unity means

that the antagonist is competitive. When one single concentration of antagonist is utilized,

the pKB value is calculated with the Gaddum Schild equation:

KB = ((CR - 1)/[antagonist])

assuming a slope equal to unity. Curve fitting was performed using PRISM 5.0 (GraphPad

Software In., San Diego, U.S.A.)

For calcium mobilization experiments pKB values were derived from inhibition

response curves using the following equation:

KB = IC50/([2 +([A]/EC50)n]1/n – 1)

where IC50 is the concentration of antagonist that produces 50% inhibition of the agonist

response, [A] is the concentration of agonist, EC50 is the concentration of agonist

producing a 50% maximal response and n is the Hill coefficient of the concentration

response curve to the agonist (Kenakin, 2004). In addition the pKB for Compound 24

evaluated at different concentrations against the concentration response curve to N/OFQ

was calculated using the following equation:

KB = [antagonist]/(slope – 1)

73

where slope is calculated from a double-reciprocal plot of equieffective concentrations of

agonist in the absence and presence of antagonist (Kenakin, 2004).

For in vivo studies, raw data from tail withdrawal experiments were converted to

the area under the time versus tail withdrawal latency curve (AUC min/s), as described by

Grisel et al. (1996). For ZP120 experiments AUC data for the time interval 0-180 min was

calculated and used for statistical analysis. For Compound 24 experiments AUC data for

the time interval (0-15 and 0-30 min for i.c.v. and i.t. studies, respectively) was used for

statistical analysis.

74

3. RESULTS & DISCUSSION

3.1 Pharmacological profile of NOP receptors coupled to calcium signalling via the

chimeric protein Gαqi5

Innovative drugs interacting selectively with the NOP receptor are under

development in several companies. However target validation studies in this field are

limited by the relative paucity of pharmacological tools which are the non-peptide agonist

Ro 64-6198 (Jenck et al., 2000) and the antagonists J-113397 (Ozaki et al., 2000) and SB-

612111 (Zaratin et al., 2004), and a few peptide ligands including the antagonist UFP-101

(Calo et al., 2002b), the partial agonists [F/G]N/OFQ(1-13)-NH2 (Guerrini et al., 1998),

Ac-RYYRWK-NH2 (Dooley et al., 1997) and ZP120 (Rizzi et al., 2002a), and the agonists

UFP-102 (Carra et al., 2005b) and UFP-112 (Rizzi et al., 2007b) (for reviews see (Calo et

al., 2005; Chiou et al., 2007; Lambert, 2008; Shoblock, 2007; Zaveri, 2003).

The identification of novel GPCR ligands are nowadays mainly based on the use of

automated fluorometers and calcium dyes. To extend this approach to Gi coupled receptors

several strategies have been developed including the use of chimeric G proteins (Conklin

et al., 1993; Kostenis et al., 2005). This strategy has been validated with a large panel of Gi

coupled receptors (Kostenis et al., 2005) including classical opioid and the NOP receptor

(Coward et al., 1999). However, in the study by Coward et al. (1999) only N/OFQ has

been used as NOP agonist and no receptor antagonists were tested. Thus, in the present

study we performed a detailed characterization of the pharmacological profile of NOP

receptors coupled to calcium signalling via the chimeric protein Gαqi5 using a large panel

of full and partial agonists as well as pure antagonists.

Results

Figure 21 (left panel) displays the response in terms of calcium mobilization of

CHOhNOP cells expressing the Gαqi5 protein to increasing concentrations of N/OFQ: the

peptide evoked immediate calcium transients in a concentration-dependent manner

displaying high potency (pEC50 ≈ 9.5) and maximal effects (≈ 200% over the basal values).

N/OFQ was found completely inactive up to 1 µM concentrations in wild type CHO stably

expressing the Gαqi5 protein. In CHOhNOP cells (not expressing the Gαqi5 protein) N/OFQ

produced a modest stimulatory effect (≈ 50% over the basal values) only at high

75

concentrations i.e. 0.1 and 1 µM. On the contrary ATP produced superimposable

concentration response curves (pEC50 ≈ 6, Emax ≈ 250%) in the three cell lines (i.e.

CHOhNOP, CHO-Gαqi5, and CHOhNOP-Gαqi5).

0 20 40 60 80 100 1200

1020304050607080

1 µM

0.1 µM

0.01 µM

1 nM

0.1 nM

0.01 nM

1 pM

buffer

N/OFQ

Time(seconds)

FIU

(x 1

03 )

0 20 40 60 80 100 1200

1020304050607080

1 µM

0.1 µM

0.01 µM

1 nM

0.1 nM

0.01 nM

1 pM

buffer

UFP-112

Time(seconds)

FIU

(x 1

03 )

Figure 21. Raw data from a single representative experiment of the concentration response curve to N/OFQ (left panel) and to UFP-112 (right panel) in calcium mobilization experiments performed in CHOhNOP cells stably expressing the Gαqi5 protein.

In order to perform a detailed characterization of NOP receptors coupled to calcium

signalling via the chimeric protein Gαqi5 a large panel of NOP receptor agonists, partial

agonists and antagonists were tested. The pharmacological activities of all these ligands are

summarized in Table 3 in terms of pEC50 and Emax values for full and partial agonists and

pKB values for antagonists.

76

Table 3. Effects of N/OFQ and NOP receptor ligands in CHO cells coexpressing the Gαqi5 chimeric protein and the human NOP receptor.

Agonist Antagonist

pEC50 (CL95%) Emax ± s.e.m. pKB (CL95%)

N/OFQ 9.54 (9.27-9.81) 167 ± 12% -

N/OFQ(1-13)-NH2 9.30 (8.84-9.76) 161 ± 12% -

UFP-112 9.05 (8.39-9.71) 173 ± 29% -

Ro 64-6198 7.98 (7.45-8.51) 178 ± 24% -

NNC 63-0532 6.80 (6.60-7.00) 111 ± 23% -

[F/G]N/OFQ(1-13)-NH2 8.03 (7.45-8.61) 91 ± 16% -

Ac-RYYRWK-NH2 8.68 (8.07-9.29) 97 ± 34% -

Ac-RYYRIK-NH2 8.16 (7.80-8.52) 99 ± 22% -

Ac-RYYRIK-ol 8.05 (7.45-8.65) 170± 23% -

UFP-113 7.97 (7.38-8.56) 104 ± 29% -

ZP120 7.15 (6.60-7.70) 97 ± 26% -

UFP-101 Inactive 7.66 (7.06-8.26)

Trap-101 Inactive 7.93 (7.39-8.47)

J-113397 Inactive 7.88 (7.43-8.33)

[Nphe1]N/OFQ(1-13)-NH2 Inactive 6.29 (5.89-6.75)

SB-612111 Inactive 8.16 (7.55-8.77)

Naloxone Inactive Inactive

Data are means ± s.e.m. of 4 separate experiments made in duplicate. Inactive: inactive up to 10 µM.

As shown in Figure 22, concentration response curves to NOP receptor agonists

were performed and compared to that to N/OFQ. All the agonists tested evoked immediate

calcium transients similar to those stimulated by N/OFQ (see as an example the raw data

relative to an UFP-112 experiment in Figure 21 right panel). The peptides N/OFQ(1-13)-

NH2 and UFP-112 mimicked the effects of N/OFQ showing similar potency and maximal

77

effects as the natural peptide. The non-peptide ligands Ro 64-6198 and NNC 63-0532

stimulated calcium levels in a concentration-dependent manner with maximal effects

similar to those of N/OFQ but approximately 30 and 500 fold lower potency.

6789101112

0

50

100

150

200N/OFQUFP-112

-Log [Agonist]

FIU

(% o

ver

the

base

line)

6789101112

0

50

100

150

200 N/OFQRo 64-6198

-Log [Agonist]

FIU

(% o

ver

the

base

line)

56789101112

0

50

100

150

200 N/OFQ

NNC 63-0532

-Log [Agonist]

FIU

(% o

ver

the

base

line)

6789101112

0

50

100

150

200 N/OFQ

N/OFQ(1-13)-NH2

-Log [Agonist]

FIU

(% o

ver

the

base

line)

Figure 22. Concentration response curve to NOP receptor agonists in calcium mobilization experiments performed in CHOhNOP cells stably expressing the Gαqi5 protein. Ligand effects were expressed as % over the baseline. Data are the mean of 4 separate experiments performed in duplicate.

Under the same experimental conditions, the pharmacological profile of a series of

NOP receptor partial agonists was investigated (Figure 23). Although the maximal effects

elicited by these peptides were lower that those of N/OFQ these differences did not reach

statistical significance (see the comparison of N/OFQ and NOP ligands maximal effects

obtained in parallel experiments showed in Figure 22). Ac-RYYRWK-NH2, Ac-RYYRIK-

NH2, Ac-RYYRIK-ol, [F/G]N/OFQ(1-13)-NH2, and UFP-113 displayed approximately 30

78

fold lower potency than N/OFQ. ZP120 was found to be the weaker compound of the

series; in fact, ZP120 was able to stimulate calcium levels only at very high concentrations

i.e. 100 and 1000 nM.

6789101112

0

50

100

150

200 N/OFQ[F/G]N/OFQ(1-13)NH2

-Log[Agonist]

FIU

(% o

ver

the

base

line)

6789101112

0

50

100

150

200N/OFQAc-RYYRWK-NH2

-Log[Agonist]

FIU

(% o

ver

the

base

line)

6789101112

0

50

100

150

200N/OFQAc-RYYRIK-NH2

-Log[Agonist]

FIU

(% o

ver

the

base

line)

6789101112

0

50

100

150

200N/OFQZP120

-Log[Agonist]

FIU

(% o

ver

the

base

line)

6789101112

0

50

100

150

200 N/OFQUFP-113

-Log[Agonist]

FIU

(% o

ver

the

base

line)

6789101112

0

50

100

150

200N/OFQAc-RYYRIK-ol

-Log[Agonist]

FIU

(% o

ver

the

base

line)

Figure 23. Concentration response curve to NOP receptor partial agonists in calcium mobilization experiments performed in CHOhNOP cells stably expressing the Gαqi5 protein. Ligand effects were expressed as % over the baseline. Data are the mean of 4 separate experiments performed in duplicate.

79

The effects of a panel of peptide and non-peptide antagonists have been also

evaluated. Inhibition response experiments were performed by testing increasing

concentrations of antagonists (10 pM – 10 µM) against a fixed concentration of N/OFQ

(10 nM), approximately corresponding to the EC80.

567891011

0

50

100

150

200

-Log[UFP-101]

FIU

(% o

ver

the

base

line)

567891011

0

50

100

150

200

-Log[Trap-101]

FIU

(% o

ver

the

base

line)

567891011

0

50

100

150

200

-Log[J-113397]

FIU

(% o

ver

the

base

line)

567891011

0

50

100

150

200

-Log[[Nphe1]N/OFQ(1-13)NH2]

FIU

(% o

ver

the

base

line)

567891011

0

50

100

150

200

-Log[SB-612111]

FIU

(% o

ver

the

base

line)

567891011

0

50

100

150

200

-Log[Naloxone]

FIU

(% o

ver

the

base

line)

Figure 24. Inhibition experiments obtained by challenging 10 nM N/OFQ with increasing concentrations of NOP receptor antagonists in the calcium mobilization assay performed in CHOhNOP cells stably expressing the Gαqi5 protein. N/OFQ effects were expressed as % over the baseline. Data are the mean of 4 separate experiments performed in duplicate.

80

As shown in Figure 24, UFP-101, J-113397 and Trap-101 were able to inhibit in a

concentration manner the stimulatory effect of 10 nM N/OFQ, showing similar pIC50

values. pKB values of 7.66, 7.88 and 7.93 were calculated from these experiments for UFP-

101, J-113397 and Trap-101, respectively. [Nphe1]N/OFQ(1-13)-NH2 behaved as a low

potency antagonist since it was able to counteract the effect of N/OFQ only at the higher

concentration tested, i.e. 10 µM. Assuming a complete inhibition for higher concentrations,

a pIC50 of 4.92 was calculated for [Nphe1]N/OFQ(1-13)-NH2 which corresponds to a pKB

value of 6.29. SB-612111 was the most potent antagonist, with a pIC50 of 6.69 and a pKB

of 8.16. Naloxone, up to 10 µM, did not modify the actions of 10 nM N/OFQ.

Finally, a Schild analysis has been performed for the compound J-113397 which

has been tested at the concentrations of 0.1, 1 and 10 µM against the effects of increasing

concentrations of N/OFQ. As showed in Figure 25, J-113397 produced a rightward shift of

the concentration response curve to N/OFQ in a concentration-dependent manner without

modifying the maximal effect elicited by the agonist. The corresponding Schild plot was

linear with a slope value not significantly different from the unity. The calculated pA2

value, 7.32, is close to the potency value (7.88) obtained in inhibition response

experiments.

56789101112

0

50

100

150

200

250

300ControlJ-113397 0.1 µMJ-113397 1 µMJ-113397 10 µM

-log[N/OFQ]

FIU(

% o

ver t

he b

asel

ine)

5670

1

2

3

pA2 = 7.32Slope = 0.99 ± 0.06

-Log[J-113397]

Log(

CR

-1)

Figure 25. Concentration-response curve to N/OFQ obtained in the absence (control) and in presence of increasing concentrations of J-113397 (0.1-10 µM) (left panel); the corresponding Schild plot is shown in the right panel. Data are the mean of 4 separate experiments performed in duplicate.

81

Discussion

The results of the present study confirmed previous findings (Coward et al., 1999)

demonstrating that it is possible to use the Gαqi5 chimeric protein to force NOP receptors

to signal via the calcium pathway. Moreover, the detailed pharmacological characterization

of NOP receptors artificially coupled to calcium signalling demonstrated that this strategy

is not associated with major distortions of the pharmacological profile of this receptor.

However some important pharmacological differences were demonstrated comparing the

present data with those from literature for a subset of partial or full agonists characterized

by slow kinetics of interaction with the NOP receptor.

In CHOhNOP, CHO-Gαqi5, and CHOhNOP-Gαqi5 cell lines ATP stimulated calcium

mobilization generating superimposable concentration response curves. This demonstrates

that the expression of the chimeric protein does not interfere with normal calcium

signalling. On the contrary, N/OFQ consistently stimulated calcium mobilization only in

the CHOhNOP-Gαqi5 cells, indicating that this response derives from the ability of the

chimeric protein to force the NOP receptor to signal via calcium. The efficiency of this

artificial coupling, measured in terms of N/OFQ Emax, indicated a signal to noise ratio in

the range 2.5 – 3. Although details regarding this point are not presented in the (Coward et

al., 1999) paper, the analysis of the results reported in Figure 2 of that article suggests a

similar efficiency. Thus the calcium mobilization response to N/OFQ in CHOhNOP-Gαqi5

cells is large and robust enough for performing both agonist and antagonist protocols.

To comprehensively characterize NOP receptors coupled to calcium signalling full

and partial agonists as well as pure antagonists were used. As far as antagonists are

concerned, all the compounds tested were found to be inactive per se up to 10 µM while

they inhibited in a concentration-dependent manner the effect evoked by 10 nM N/OFQ.

The following order of potency of antagonist has been recorded:

SB-612111 > J-113397 = Trap-101 > UFP-101 > [Nphe1]N/OFQ(1-13)-NH2

(naloxone inactive). These results are superimposable to those obtained by testing the

antagonists in [35S]GTPγS binding experiments performed on CHOhNOP cell membranes

(McDonald et al., 2003b; Spagnolo et al., 2007; Trapella et al., 2006) as well as in mouse

vas deferens bioassay studies (Bigoni et al., 2000a; Calo et al., 2000a; Calo et al., 2002b;

Spagnolo et al., 2007; Trapella et al., 2006). The correlation analysis of the present data

with those previously obtained in the mouse vas deferens assay (data taken from the quoted

articles) yielded a determination coefficient r2 of 0.90. Therefore it can be proposed that at

least for ligand devoid of efficacy (i.e. pure antagonists), the Gαqi5 NOP receptor calcium

82

assay generates results superimposable to those obtained with classical techniques useful

for studying Gi coupled receptors. This applies not only to antagonist potency but also to

the type of antagonism as demonstrated, at least for J-113397, by the results of the Schild

analysis. In fact a competitive type of NOP antagonism was demonstrated for J-113397

both by the present study and by previous investigations performed in the mouse vas

deferens preparation (Bigoni et al., 2000a).

All the NOP receptor full agonists evaluated in the present study were able to evoke

maximal effects similar to those obtained with N/OFQ with the following order of potency:

N/OFQ = N/OFQ(1-13)-NH2 > UFP-112 > Ro 64-6198 > NNC 63-0532

These results are only partially in agreement with findings from literature. In fact,

data regarding N/OFQ, N/OFQ(1-13)-NH2 and NNC 63-0532 perfectly match those

obtained with [35S]GTPγS binding (McDonald et al., 2003b; Thomsen et al., 2000b) and

mouse vas deferens and guinea pig ileum assays (Calo et al., 1996; Guerrini et al., 2004).

On the contrary, in the same assays Ro 64-6198 displayed potency values similar to those

of N/OFQ (Hashiba et al., 2002b; Jenck et al., 2000; Rizzi et al., 2001c) while UFP-112

was found to be 30 fold more potent than the naturally occurring peptide (Arduin et al.,

2007; Rizzi et al., 2007b). Thus, the potency of both Ro 64-6198 and UFP-112 is

significantly lower in the Gαqi5 NOP receptor calcium assay than in other assays. The

interpretation of these discrepant results is far from obvious. The chemical nature of the

small molecule Ro 64-6198 and that of the N/OFQ analogue UFP-112 is very different

suggesting that chemical features are not relevant. It is worthy of note that isolated tissue

experiments demonstrated an important characteristic which is common to UFP-112 and

Ro 64-6198. Indeed, as described in detail in previous publications (Arduin et al., 2007;

Rizzi et al., 2007b), the kinetics of the inhibitory effect elicited by N/OFQ on the

electrically induced twitch response is rapid and immediately and completely reversible

after washing while that of both UFP-112 and Ro 64-6198 is characterized by slow onset,

and slow and partial reversibility after washing. The slow kinetics of action of these

ligands may be relevant for the estimation of their potency in the Gαqi5 NOP receptor

calcium assay. In fact, the long time required to obtain full activation of NOP receptors

with these agonists may be incompatible with the rapid kinetics which characterized the

calcium transient response. The different kinetics of N/OFQ and UFP-112 recorded in

isolated tissues could not be detected in the present calcium mobilization experiments (see

Figure 21). However, we cannot exclude that the relative low potency displayed by UFP-

83

112 and Ro 64-6198 in the calcium assay merely depends on subtle changes of the receptor

pharmacological profile induced by the chimeric protein.

Results obtained with the panel of NOP partial agonists are different from those

described in the literature in terms of ligand efficacy and, for a subset of compounds,

ligand potency. All the ligands evaluated in the present study induced maximal effects

lower than that of the natural peptide N/OFQ although these differences did not reach

statistical significance. Thus ligand efficacy estimated in the Gαqi5 NOP receptor calcium

assay is higher than in other assays. However this result is not completely unexpected.

NOP receptor partial agonists displayed consistent efficacy often behaving as full agonists

in the cAMP assay (Butour et al., 1998; Dooley et al., 1997; Kapusta et al., 2005b; Mason

et al., 2001) while the same compounds showed limited and sometimes negligible efficacy

in [35S]GTPγS binding studies (Berger et al., 2000b; Dooley et al., 1997; Kocsis et al.,

2004; Mason et al., 2001). Thus, the estimated efficacy of this kind of ligand strongly

depends on the efficiency of the stimulus-response coupling which is different in the

different assays. Those tests in which signal amplification phenomena make the efficiency

of the stimulus-response coupling high (i.e. cAMP and calcium assays) tend to

overestimate efficacy while those tests in which there is no amplification and the efficiency

of the stimulus-response coupling is low (i.e. [35S]GTPγS binding) tend to underestimate

efficacy. This concept has been experimentally validated in the study by McDonald et al.

(2003a) where stimulus-response coupling of both the cAMP and [35S]GTPγS binding

assays was modulated by changing the number of membrane receptors using a NOP

inducible expression system. In this study partial agonist efficacy could be manipulated to

encompass full and partial agonism along with pure antagonism (McDonald et al., 2003a).

In the present study the partial agonist order of potency was as follows: Ac-

RYYRWK-NH2 > Ac-RYYRIK-NH2 = Ac-RYYRIK-ol = [F/G]N/OFQ(1-13)-NH2 =

UFP-113 > ZP120. Similarly to that found with full agonists these results are only partially

in line with literature findings. Indeed, the following order of potency, which is in good

agreement with the present results, has been described using classical tests for Gi coupled

receptors: Ac-RYYRWK-NH2 > Ac-RYYRIK-NH2 = Ac-RYYRIK-ol > [F/G]N/OFQ(1-

13)-NH2 (Dooley et al., 1997; Gunduz et al., 2006; Kocsis et al., 2004; Mason et al.,

2001). In contrast, UFP-113 (Arduin et al., 2007) and ZP120 (Kapusta et al., 2005b; Rizzi

et al., 2002a) displayed potency values 10-30 fold higher than their templates

([F/G]N/OFQ(1-13)-NH2 and Ac-RYYRWK-NH2 respectively). Interestingly, in the

84

electrically stimulated mouse vas deferens ZP120 showed kinetics of action comparable to

those described for Ro 64-6198 and UFP-112 (inhibitory effects slow to develop and

slowly and only partially reversible after washing (Rizzi et al., 2002a). This kind of

evidence is not available for UFP-113 because this peptide produced variable agonist

effects mainly behaving as a NOP antagonist in the mouse vas deferens (Arduin et al.,

2007). However, a similar type of kinetics of action can be hypothesized for UFP-113

based on its close structural similarities with UFP-112 (Arduin et al., 2007). Therefore, as

proposed for the NOP full agonists Ro 64-6198 and UFP-112, the relatively low potency of

UFP-113 and ZP120 in the Gαqi5 NOP receptor calcium assay may be the consequence of

the long time required to activate the NOP receptor with these ligands which is not

compatible with the rapid kinetics of the calcium transient response.

In conclusion, the present study confirmed and extended previous findings

demonstrating that the chimeric protein Gαqi5 is indeed capable of coupling to NOP

receptors to the Ca2+ signalling pathway. Moreover, results obtained from the detailed

pharmacological characterization of the NOP receptor with the Gαqi5 calcium assay

demonstrated that this approach represents a very useful strategy for the screening of NOP

receptor antagonists. Results obtained with this class of ligands in the Gαqi5 NOP receptor

calcium assay are superimposable to those collected with Gi based biochemical assays

performed at recombinant or native NOP receptors or with bioassays performed on isolated

tissues expressing native NOP receptors. In contrast, the usefulness of this assay for the

screening of partial and full agonists appears to be limited by its tendency to estimate high

efficacy for partial agonists and low potency for partial and full agonists characterized by

slow kinetics of action.

3.2 Further studies on the pharmacological features of the nociceptin/orphanin FQ

receptor ligand ZP120

ZP120 is a NOP receptor ligand (Larsen et al., 2001) which has been generated by

applying the structure inducing probes technology (Larsen, 1999) to the NOP selective

partial agonist Ac-RYYRWK-NH2 (Dooley et al., 1997) with the aim of improving its

metabolic stability and potency without modifying its pharmacodynamic properties. This

85

strategy appeared to be successful since ZP120 has been demonstrated in in vitro

experiments to bind to the NOP receptor with high affinity, to inhibit forskolin induced

cAMP accumulation in HEK293hNOP cells and electrically induced contractions of the

mouse vas deferens showing compared to N/OFQ higher potency and lower maximal

effects (Kapusta et al., 2005b; Rizzi et al., 2002a). Furthermore in in vivo experiments

ZP120 mimicked N/OFQ actions including the pronociceptive and locomotor inhibitory

effects after supraspinal administration (Rizzi et al., 2002a) and the diuretic effects after

intravenous administration (Kapusta et al., 2005b), showing a longer duration of action.

Interestingly, unlike N/OFQ, ZP120 did not modify per se cardiovascular parameters but

rather antagonized the bradycardic and hypotensive action of N/OFQ (Kapusta et al.,

2005b). This confirmed the findings obtained with other NOP receptor partial agonists i.e.

[F/G]N/OFQ(1-13)-NH2, Ac-RYYRWK-NH2 and Ac-RYYRIK-NH2 and corroborate the

proposal that NOP receptor partial agonists produce functionally selective effects on

cardiovascular and renal functions ranging from full agonist (water diuresis) to antagonist

(bradycardia and hypotension) action (Kapusta et al., 2005a).

As far as the ZP120 receptor mechanism is concerned, we have previously

reported that the effects of this peptide in the mouse vas deferens assay are resistant to

naloxone while sensitive to the NOP selective antagonist J-113397 (Ozaki et al., 2000)

which displayed similar pA2 values against ZP120 (7.80) and N/OFQ (7.81) (Rizzi et al.,

2002a). Recently, it has been reported that N/OFQ and ZP120 are both able to inhibit in a

concentration-dependent manner the contractions evoked by electrical field stimulation in

rat mesenteric arteries (Simonsen et al., 2008). However, in this preparation, the NOP

selective peptide antagonist UFP-101 (Calo et al., 2005; Calo et al., 2002b) antagonized

the action of N/OFQ but not that of ZP120 (Simonsen et al., 2008). Several important

differences in terms of molecules (J-113397 vs UFP-101), preparations (vas deferens vs

mesenteric arteries), and species (mouse vs rat) used in the two above mentioned studies

do not allow an easy interpretation of these contrasting findings. Therefore the aim of the

present study was to extensively investigate the pharmacological features of ZP120 by

comparing its effects with N/OFQ in the mouse and rat vas deferens and by challenging

the actions of these NOP agonists with both J-113397 and UFP-101. Moreover the in vitro

(inhibition of electrically stimulated vas deferens) and in vivo (supraspinal pronociceptive

effect) actions of ZP120 were reassessed in tissues taken from and in mice knockout for

the NOP receptor gene, respectively.

86

Results

Electrically stimulated isolated tissues - The NOP receptor ligands N/OFQ and ZP120

inhibited the electrically induced contraction of the mouse and rat vas deferens in a

concentration-dependent manner (Figure 26). N/OFQ displayed similar values of potency

(pEC50 ≈ 7.3) and maximal effects (≈ 80% inhibition of control twitch) in mouse and rat

tissues. ZP120 (pEC50 ≈ 8.8) was about 30 fold more potent than N/OFQ but produced

maximal effects (≈ 60% inhibition of control twitch) significantly lower than those of the

natural peptide in both preparations.

Mouse Vas Deferens

-12 -11 -10 -9 -8 -7 -6 -50

20

40

60

80

100

N/OFQZP120

*

Log[Ligand]

% C

ontr

ol T

witc

h

Rat Vas Deferens

-12 -11 -10 -9 -8 -7 -6 -5 -40

20

40

60

80

100

N/OFQZP120

*

Log[Ligand]

% C

ontro

l Tw

itch

Figure 26. Concentration response curves to N/OFQ and ZP120 in the electrically stimulated mouse (left panel) and rat (right panel) vas deferens. Data are the mean ± s.e.m. of 5 separate experiments. *p<0.05 vs N/OFQ, according to the Student t-test.

In addition and confirming previous findings obtained in the mouse vas deferens

(Rizzi et al., 2002a), the kinetics of the inhibitory effects elicited by ZP120 were different

from that of N/OFQ in both preparations. The action of the natural peptide took place

immediately after adding the peptide to the bath, was rapidly reversible after washing, and

could be repeated in the same tissue, on the contrary, the effects of ZP120 were slow to

develop, slowly reversible after washing, and could not be repeated in the same tissue.

Typical tracings showing the concentration response-curve to N/OFQ and ZP120 in the rat

vas deferens are displayed in Figure 27.

87

Figure 27. Typical tracings showing the concentration response curve to N/OFQ (0.1 nM-10 µM, left panel) and ZP120 (10 pM-100 nM; right panel) in the electrically stimulated rat vas deferens.

As shown in the Figure 28, the effects of N/OFQ and ZP120 were evaluated in the

presence of the NOP selective antagonists UFP-101 (1 µM) and J-113397 (100 nM) in

both preparations.

Con

trol

twitc

h

5 min

WW

N/OFQ ZP120

: Drugs injectionW: Wash out

Con

trol

twitc

h

5 min

WW

N/OFQ ZP120

: Drugs injectionW: Wash out5 min

WW

N/OFQ ZP120

: Drugs injectionW: Wash out

88

-12 -11 -10 -9 -8 -7 -6 -5 -40

20

40

60

80

100

ControlUFP-101 1 µMJ-113397 100 nM

Log[N/OFQ]

Con

trol T

witc

h %

-12 -11 -10 -9 -8 -7 -60

20

40

60

80

100


Log[ZP120]

Cont

rol T

witc

h %

-11 -10 -9 -8 -7 -6 -5 -40

20

40

60

80

100


Log[N/OFQ]

Cont

rol T

witc

h %

-12 -11 -10 -9 -8 -7 -60

20

40

60

80

100


Log[ZP120]

Cont

rol T

witc

h %

Mouse Vas Deferens

Rat Vas Deferens

Figure 28. Concentration-response curves to N/OFQ (left panels) and ZP120 (right panels) measured in the absence and in presence of UFP-101 (1 µM) and J-113397 (100 nM) in the electrically stimulated mouse (top panels) and rat (bottom panels) vas deferens. Data are the mean ± s.e.m. of 4 separate experiments.

Both the antagonists did not modify per se the control twitches. In the mouse and

rat vas deferens J-113397 produced a rightward shift of the concentration-response curve

to N/OFQ without significantly affecting the maximal agonist response. J-113397 pKB

values derived from these experiments were 7.69 and 8.04, respectively. Superimposable

results were obtained by challenging J-113397 vs ZP120. J-113397 competitively

antagonized ZP120 effects in the mouse and rat tissues with the following pKB values: 7.76

and 7.89. In the mouse and rat vas deferens the NOP selective antagonist UFP-101

displaced to the right the concentration-response curve to N/OFQ yielding pKB values of

89

7.37 and 6.79, respectively. UFP-101 did not modify the inhibitory effect of ZP120 in both

preparations. Antagonist pKB values obtained from these experiments are summarized in

Table 4.

Table 4. pKB values of UFP-101 and J-113397 vs N/OFQ and ZP120 in the electrically stimulated mouse and rat vas deferens. Data are mean of 4 separate experiments. CL95%: confidence limit.

Mouse Vas Deferens Rat Vas Deferens pKB (CL95%) pKB (CL95%) UFP-101 J-113397 UFP-101 J-113397

N/OFQ 7.37 (7.13-7.61)

7.69 (7.17-8.22)

6.79 (6.45-7.13)

8.04 (7.54-8.54)

ZP120 <6 7.76 (7.67-7.85) <6 7.89

(7.02-8.76)

The effects of N/OFQ and ZP120 and those elicited by the selective DOP agonist

DPDPE, were investigated in the electrically stimulated mouse vas deferens taken from

NOP(+/+) and NOP(-/-) mice (Figure 29).

-10 -9 -8 -7 -60

25

50

75

100

NOP(+/+)NOP(-/-)

Log[N/OFQ]

% C

ontr

ol T

witc

h

-11 -10 -9 -8 -7 -60

25

50

75

100

NOP(+/+)NOP(-/-)

Log[ZP120]

% C

ontro

l Tw

itch

-11 -10 -9 -8 -7 -60

25

50

75

100

NOP(+/+)NOP(-/-)

Log[DPDPE]

% C

ontro

l Tw

itch

Figure 29. Electrically stimulated mouse vas deferens. Concentration response curve to N/OFQ, ZP120 and DPDPE in tissues taken from NOP(+/+) and NOP(-/-) mice. Data are the mean ± s.e.m. of 4 separate experiments.

In NOP(+/+) tissues, ZP120 mimicked the inhibitory effect of N/OFQ (pEC50 7.62;

Emax 91 ± 1%) showing higher potency (pEC50 9.10) but lower maximal effects (Emax 73 ±

3%). In tissues taken from NOP(-/-) mice, N/OFQ was found inactive and ZP120 slightly

90

inhibited the electrically induced contraction only at the highest concentration tested (0.3

µM). In parallel experiments, the DOP receptor selective agonist DPDPE displayed similar

potency (pEC50 8.40 and 8.20) and maximal effects (Emax 93 ± 3% and 91 ± 5%) in tissues

taken from NOP(+/+) and NOP(-/-) mice, respectively. The values of potency and maximal

effects derived from concentration response curves to N/OFQ and ZP120 in the electrically

stimulated vas deferens taken from rats and mice (Swiss, NOP(+/+) and NOP(-/-)) are

summarized in Table 5.

Table 5. Effects of N/OFQ and ZP120 in the electrically stimulated mouse and rat vas deferens. Data are mean of 5 separate experiments. CL95%: confidence limit. ND: could not be determined. *p<0.05 vs N/OFQ, according to the Student t-test. Mouse Vas Deferens Rat Vas

Deferens Swiss mice NOP(+/+) mice NOP(-/-) mice pEC50

(CL95%) Emax ± s.e.m.

pEC50 (CL95%)

Emax ± s.e.m.

pEC50 (CL95%)

Emax ± s.e.m. pEC50

(CL95%) Emax ± s.e.m.

N/OFQ 7.37 (7.27-7.47) 80 ± 2% 7.62

(7.56-7.68) 91 ± 1% < 6 ND 7.25 (7.08-7.42) 83 ± 2%

ZP120 8.78 (8.61-8.95) 56 ± 3%* 9.10

(8.88-9.32) 73 ± 3%* < 6 ND 8.80 (8.41-9.19) 64 ± 4%*

Mouse tail withdrawal assay - NOP(+/+) mice injected i.c.v. with saline did not show any

modification of gross behaviour. Those injected with high doses of N/OFQ (10 nmol) or

ZP120 (1 nmol) displayed a clear decrease in locomotor activity and muscular tone, ataxia,

and partial loss of the righting reflex. These actions on mice gross behaviour were

previously reported in Swiss mice both for N/OFQ (Calo et al., 1998b) and ZP120 (Rizzi

et al., 2002a). The i.c.v. injection of these peptides in NOP(-/-) mice did not produce any

obvious modifications of their spontaneous behaviour.

Results summarized in Figure 30 (left panel) show that baseline tail withdrawal

latency of NOP(+/+) mice was 5.04 ± 0.73 s. In animals treated with saline this parameter

was stable around 5-6 s over the time course of the experiment. The i.c.v. administration of

10 nmol N/OFQ produced an immediate (peak effect at 5 min) pronociceptive action

which lasted for approx 1 h. The injection of 1 nmol ZP120 mimicked the pronociceptive

effect of N/OFQ, however it was characterized by a slow onset of action (peak effect at

120-180 min) and was longer lasting with the reduction of tail withdrawal latency still

evident after 3h from peptide injection. This pattern of effects of N/OFQ and ZP120 is

91

superimposable to that previously reported in Swiss mice (Calo et al., 1998b; Rizzi et al.,

2002a).

In line with previous findings (Nishi et al., 1997), baseline latency of NOP(-/-)

mice (5.21 ± 0.90 s) was similar to that of NOP(+/+) mice. The injection of saline in

NOP(-/-) animals produced a slight increase in tail-withdrawal latency which was then

stable over the time course of the experiment (Figure 30, right panel). In NOP(-/-) mice,

the i.c.v. injection of both N/OFQ and ZP120 did not produce any effect with tail

withdrawal latencies superimposable to that of saline treated animals (Figure 30, right

panel).

0 30 60 90 120 150 1800

3

6

9

12 SalineN/OFQ 10 nmol

NOP(+/+)

ZP120 1 nmol

**

*

Time (min)

TW L

aten

cy (s

)

0 30 60 90 120 150 1800

3

6

9

12 SalineN/OFQ 10 nmol

NOP(-/-)

ZP120 1 nmol

Time (min)

TW L

aten

cy (s

)

Figure 30. Effects of 10 nmol N/OFQ and 1 nmol ZP120 given i.c.v. in the tail withdrawal assay performed in NOP(+/+) (left panel) and NOP(-/-) (right panel) mice. Data are the mean ± s.e.m. of 4 separate experiments. *p<0.05 and **p<0.01 versus saline according to ANOVA followed by the Dunnett's test.

Discussion

This study provides novel evidence that confirms and extends our knowledge of the

pharmacological profile of ZP120 in rodents. This peptide behaves as a selective NOP

receptor partial agonist and displays kinetics of action characterized by slow onset and

long lasting effects. The antagonist sensitivity of ZP120 effects (J-113397 sensitive,

naloxone and UFP-101 resistant) is not superimposable to that of the endogenous NOP

ligand N/OFQ (J-113397 and UFP-101 sensitive, naloxone resistant). However the

exclusive involvement of the NOP receptor protein in the in vitro as well as in vivo effects

of ZP120 (and N/OFQ) was clearly demonstrated by knockout studies. Therefore the

92

different sensitivity of N/OFQ and ZP120 to UFP-101 can be attributed to their diverse

way of binding to and activating the NOP receptor.

The present data obtained in parallel experiments performed on mouse and rat

tissues confirmed previous findings (Kapusta et al., 2005b; Rizzi et al., 2002a; Simonsen

et al., 2008) demonstrating that ZP120 behaves as a NOP receptor partial agonist. The

following evidence and considerations make this pharmacological feature of ZP120

extremely important. Partial agonists may discriminate between the different

pharmacological effects evoked by full agonists in different systems/organs depending on

the efficiency of the stimulus-response coupling that characterize each of the

systems/organs (Kenakin, 2004). This has been demonstrated for the cardiovascular and

renal effects of NOP ligands. I.v. administration of the full agonist N/OFQ produce in

rodents cardiovascular inhibitory effects (i.e. hypotension and bradycardia) associated with

diuretic, in particular aquaretic, effects (Kapusta, 2000). In contrast, i.v. administration of

NOP receptor partial agonists ([F/G]N/OFQ(1-13)-NH2, Ac-RYYRWK-NH2 and Ac-

RYYRIK-NH2) only mimicked the renal effects of N/OFQ without modifying

cardiovascular parameters (Kapusta, 2000). This same pattern of effects was demonstrated

for ZP120 (Kapusta et al., 2005b). Therefore in water-retaining states with hyponatremia

such as heart failure, NOP receptor partial agonists like ZP120 represent a better

therapeutic option than full agonists. In addition to partial agonist behaviour, ZP120

displays slow onset, long lasting effects with high potency. These features have been

confirmed in the present study both in vitro and in vivo and are in line with that reported in

the literature. ZP120 has been shown to mimic with higher potency and longer lasting

effects the following N/OFQ actions: inhibition of locomotor activity and pronociceptive

effects after supraspinal administration in mice (Rizzi et al., 2002a) and sodium-sparing

aquaretic activity after i.v. administration in rats (Kapusta et al., 2005b). Collectively these

pharmacological features make ZP120 a promising and innovative drug candidate for

treating heart failure (Lambert, 2008).

The receptor mechanism involved in ZP120 actions was investigated by using two

chemically unrelated and selective NOP receptor antagonists the peptide UFP-101 (Calo et

al., 2005; Calo et al., 2002b) and the non-peptide J-113397 (Ozaki et al., 2000) as well as

mice knock out for the NOP receptor gene (Nishi et al., 1997). UFP-101 and J-113397

represent standard antagonists for NOP receptor investigations with more than 50 papers

reported in the literature for each of these molecules describing their antagonist properties

vs N/OFQ (Chiou et al., 2007; Lambert, 2008). In line with this picture, in the present

93

study both molecules competitively antagonized N/OFQ effects in mouse and rat tissues

showing the expected order of potency of antagonists i.e. J-113397 (pKB ≈ 7.8) > UFP-101

(pKB ≈ 7.3). J-113397 was also found to be active against ZP120 whose effects were

antagonized in both preparations with pKB values superimposable to those obtained against

N/OFQ. In contrast, UFP-101 was found inactive against ZP120. Similar findings were

obtained in separate studies where the effects of N/OFQ and ZP120 in the mouse vas

deferens were found to be sensitive to J-113397 (Rizzi et al., 2002a) while those of N/OFQ

but not ZP120 to be sensitive to UFP-101 in rat mesenteric arteries (Simonsen et al., 2008).

Since we investigated in parallel studies both rat and mouse preparations obtaining

consistent pharmacological results in tissues from the two species, we can rule out that

species specific NOP receptor isoforms play a role in the pattern of antagonist sensitivity

of ZP120 effects. As mentioned above ZP120 differs from N/OFQ in terms of kinetics of

action and this might be relevant for antagonist sensitivity. However, other NOP selective

peptide agonists such as UFP-102 (Carra et al., 2005b) and UFP-112 (Arduin et al., 2007)

characterized by kinetics of action superimposable to that of ZP120 were found to be

sensitive to the antagonist effect of UFP-101 (Carra et al., 2005b; Rizzi et al., 2007b), thus

suggesting that the agonist kinetics of action are not important for the pattern of antagonist

sensitivity. A different receptor mechanism can be suggested to interpret the diverse

antagonist sensitivity of ZP120 and N/OFQ. However, the in vitro and in vivo knockout

studies performed in the frame of the present study clearly demonstrated that the effects of

both agonists, which are no longer evident in NOP(-/-) tissues and animals, are due to the

selective activation of the NOP receptor protein. Therefore, the most reasonable suggestion

that can be proposed to collectively interpret these findings is that by Simonsen et al.

(2008) i.e. N/OFQ and the hexapeptide Ac-RYYRWK-NH2 (from which ZP120 derives)

have distinct modes of interaction with the NOP receptor and UFP-101 is able to

discriminate between them by blocking N/OFQ binding but not that of ZP120. This

suggestion is corroborated by the following evidence: in elegant photoaffinity labelling

studies the group of Meunier investigated the NOP receptor regions that bind N/OFQ and

hexapeptides; in particular N/OFQ binds to extracellular loop III and transmembrane helix

VII (Mouledous et al., 2000) while the hexapeptides bind to transmembrane helix II (Bes

et al., 2003). However, some overlapping of the NOP receptor regions that recognized

N/OFQ and hexapeptides should be hypothesized because the binding of N/OFQ and

hexapeptides to the NOP receptor is mutually exclusive as demonstrated by receptor

binding studies (Dooley et al., 1997; Thomsen et al., 2000b) and by functional antagonist

94

studies performed in those preparations/assays in which the hexapeptides (or their

analogues) displayed small or no efficacy and where a competitive type of interaction has

been reported for these peptides. These antagonist studies were performed investigating

[35S]GTPγS binding to G proteins in membranes and sections of rat brain (Berger et al.,

1999), chronotropic effect on neonatal rat cardiomyocytes (Berger et al., 1999), the

electrically stimulated mouse vas deferens (Gunduz et al., 2006; Li et al., 2008) or the

cardiovascular parameters of the rat (Kapusta et al., 2005a). On the other hand, the NOP

receptor site recognized by UFP-101, which might be similar but not identical to that of

N/OFQ, is not overlapping with that of the hexapeptides and this may explain the lack of

effect of UFP-101 against ZP120 reported by Simonsen et al. (2008) in rat mesenteric

arteries and confirmed in the present study in vas deferens tissues from rats and mice.

In conclusion, the present investigation demonstrated via in vitro and in vivo

receptor knockout studies that ZP120 is a highly selective NOP receptor ligand. Moreover

a diverse antagonist sensitivity of N/OFQ (J-113397 and UFP-101 sensitive) and ZP120 (J-

113397 sensitive and UFP-101 resistant) which can not be explained in terms of species

specific NOP receptor isoforms, diverse agonist kinetics of action, or different receptor

mechanism, was reported. This diverse antagonist sensitivity may derive from different

mechanisms of binding to the NOP receptor for N/OFQ, ZP120, and UFP-101.

Collectively this study provides further evidence that ZP120 behaves as a selective NOP

receptor partial agonist characterized by high potency and able to evoke slow onset long

lasting effects. Collectively these pharmacological features make ZP120 not only a useful

pharmacological tool to be used in preclinical investigations but also a drug candidate for

exploiting the therapeutic benefits derived by a prolonged partial activation of peripheral

NOP receptors.

3.3 Pharmacological characterization of the nociceptin/orphanin FQ receptor non-

peptide antagonist Compound 24

A novel NOP receptor non-peptide antagonist, 1-benzyl-N-{3-[spiroisobenzofuran-

1(3H),4’-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide, has been recently identified by

Banyu investigators and named Compound 24 (Goto et al., 2006). The synthesis of this

novel ligand is relatively easy and the overall yield relatively high (25% in our laboratory,

31% in Goto et al. (2006)). This is particularly true when compared to the synthesis of

95

other non-peptide NOP antagonists such as J-113397 and SB-612111 whose synthesis is

very difficult and of low overall yield (≈ 1% for both molecules in our laboratories, C.

Trapella personal communication).

Thus, the aim of the present study was the synthesis and detailed investigation of

the pharmacological profile of Compound 24. The novel ligand was investigated in vitro in

receptor binding and [35S]GTPγS experiments performed in CHOhNOP cell membranes, in

calcium mobilization experiments performed in CHOhNOP cells expressing the Gαqi5

protein, and in N/OFQ sensitive isolated tissues. Finally, the in vivo actions of Compound

24 were assessed in mice using the tail withdrawal assay.

Results

Receptor binding assay - In receptor binding experiments performed on CHOhNOP cell

membranes Compound 24 displaced [3H]N/OFQ in a concentration-dependent manner

with subnanomolar affinity (pKi 9.62, Table 6). Under the same experimental conditions,

Compound 24 did not bind the hDOP receptor and showed low affinities for MOP and

KOP sites (pKi 6.72 and 6.47, respectively). In contrast, the universal opioid receptor

ligand naloxone did not bind the NOP receptor but displayed the expected rank order of

affinity at classical opioid receptors i.e. MOP (pKi 9.25) > KOP (pKi 8.35) > DOP (pKi

7.67) (Table 6).

Table 6. Affinities of Compound 24 and naloxone at NOP and classical opioid receptors expressed in CHO cell membranes.

receptor NOP MOP DOP KOP radioligand [3H]N/OFQ [3H]DPN [3H]DPN [3H]DPN naloxonea < 6 9.25

(9.04 – 9.46)7.67

(7.59 – 7.75) 8.35

(8.20 – 8.50)

Compound 24 9.62 (9.47 – 9.77)

6.72 (6.43 – 6.97) < 6 6.47

(6.20 – 6.74) a: naloxone affinities at classical opioid receptors are from Vergura et al. (2008). Data are mean (CL95%) of 3 separate experiments. [35S]GTPγS binding assay - In CHOhNOP cell membranes N/OFQ stimulated [35S]GTPγS

binding in a concentration-dependent manner with a pEC50 value of 8.95 ± 0.05 and Emax

96

of 11.71 ± 0.37 (Figure 31, left panel). The antagonistic properties of Compound 24 were

evaluated over the 1-100 nM concentration range, in order to obtain data for a Schild

analysis. Compound 24 up to 10 µM did not elicit any stimulation of [35S]GTPγS binding

in CHOhNOP cell membranes, however it produced a concentration-dependent and parallel

shift of the concentration response curve to N/OFQ without modifying the maximal effects

induced by the agonist (Figure 31, left panel). Schild analysis of the data (Figure 31, right

panel) demonstrated a linear (r2 = 0.95) plot with a slope not significantly different from

unity. The extrapolated pA2 value was 9.98.

-12 -11 -10 -9 -8 -7 -6 -5 -40

2

4

6

8

10

12

14 ControlCompound 24 1 nMCompound 24 10 nMCompound 24 100 nM

Log[N/OFQ]

Stim

ulat

ion

Fact

or

-9 -8 -70

1

2

3

pA2 = 9.98Slope = 1.07 ± 0.10r2 = 0.95

Log[Compound 24]

Log(

CR

-1)

Figure 31. Left panel: concentration response curves to N/OFQ obtained in the absence (control) and presence of increasing concentrations of Compound 24 (1 – 100 nM) in the [35S]GTPγS binding assay performed on CHOhNOP cell membranes. The relative Schild Plot is shown in the right panel. Data are mean ± s.e.m. of 4 separate experiments.

Calcium mobilization assay - In CHOhNOP cells stably expressing the Gαqi5 chimeric

protein N/OFQ evoked a concentration-dependent stimulation of calcium release (pEC50

9.24 (CL 95% 9.10 – 9.38)). Compound 24 was inactive per se but in the range 0.01 nM–10

µM concentration-dependently inhibited calcium mobilization induced by 10 nM N/OFQ

with a pKB value of 9.03 ± 0.20 (Table 7).

97

Table 7. Antagonist potencies of Compound 24 and naloxone evaluated in calcium mobilization experiments performed in CHO cells expressing NOP or classical opioid receptors and the Gαqi5 protein.

receptor NOP MOP DOP KOP

agonist N/OFQ 10 nM

Dermorphin 100 nM

DPDPE 100 nM

Dynorphin A 100 nM

naloxone < 6 9.09

(8.73-9.45) 7.32

(6.11-8.53) 7.14

(6.60-7.68)

Compound 24 9.03 (8.83-9.23) < 6 < 6 < 6

Data are mean (CL95%) of 4 separate experiments.

Naloxone was inactive up to 1 µM. To assess the selectively of action of

Compound 24 similar experiments were performed in CHO cells stably expressing Gαqi5

and classical opioid receptors. Dermorphin, DPDPE and dynorphin A were used in these

experiments as agonists for MOP, DOP and KOP receptors, respectively. They produced a

concentration-dependent stimulation of calcium mobilization with the following values of

pEC50 and Emax: dermorphin 7.93 (CL 95% 7.67 – 8.19), 196 ± 9%; DPDPE 8.82 (CL 95%

8.43 – 9.21), 130 ± 10%; dynorphin A 8.47 (CL 95% 8.16 – 8.78) 174 ± 14%. Naloxone

inhibited the effects of these agonists showing higher potency at MOP (pKB 9.09) than

KOP (pKB 7.14) and DOP (pKB 7.32) (Table 7). In contrast Compound 24 was inactive up

to 1 µM against DPDPE, dynorphin A, and dermorphin (Table 7).

To investigate the type of antagonism produced by Compound 24 in calcium

mobilization experiments a Schild analysis has been performed by testing this molecule at

various concentrations (0.01, 0.1 and 1 µM) against the concentration-response curve to

N/OFQ. As shown in Figure 32, Compound 24 produced a rightward shift of the

concentration-response curve to N/OFQ in a concentration-dependent manner. However,

the maximal effects elicited by N/OFQ appear to be slightly but significantly reduced by

Compound 24. A pKB value of 8.73 was derived from these experiments. It is worthy of

note that this value is close to that obtained in inhibition response experiments (9.03).

98

56789101112-50

0

50

100

150

200ControlCompound 24 10 nMCompound 24 100 nMCompound 24 1 µM

*

-Log[N/OFQ]

FIU

(% o

ver t

he b

asel

ine)

Figure 32. Concentration response curves to N/OFQ obtained in the absence (control) and presence of increasing concentrations of Compound 24 (10 nM - 1 µM) in the calcium mobilization assay performed in CHOhNOP cells stably expressing the Gαqi5 protein. Data are mean ± s.e.m. of 4 separate experiments performed in duplicate. *p<0.05 versus control according to ANOVA followed by Dunnett’s test.

Electrically stimulated isolated tissues - Compound 24 was assessed against N/OFQ in the

electrically stimulated mouse and rat vas deferens and guinea pig ileum. In the mouse vas

deferens N/OFQ inhibited the twitch response to electrical field stimulation in a

concentration-dependent manner (pEC50 value 7.46, Figure 33 left panel). Compound 24,

tested over the concentration range 10 nM -1 µM, did not modify per se the electrically-

induced twitches, but displaced to the right the concentration response curve to N/OFQ in a

concentration-dependent manner. Curves obtained in the presence of Compound 24 were

parallel to the control (Figure 33, left panel) with no modification of the agonist maximal

effect. The corresponding Schild plot was linear (r2 = 1.00) with a slope not significantly

different from unity, yielding a pA2 value of 8.44 (Figure 33, right panel).

99

-10 -9 -8 -7 -6 -5 -40

20

40

60

80

100

ControlCompound 24 10 nMCompound 24 100 nMCompound 24 1 µM

Log[N/OFQ]

% C

ontro

l Tw

itch

-8 -7 -60

1

2

3 pA2= 8.44slope= 0.99 ± 0.01r2= 1

Log[Compound 24]

Log(

CR-1

)

Figure 33. Left panel: concentration response curves to N/OFQ obtained in the absence (control) and presence of increasing concentrations of Compound 24 (10 nM – 1 µM) in the electrically stimulated mouse vas deferens. The relative Schild Plot is shown in the right panel. Data are mean ± s.e.m. of 4 separate experiments.

In the rat vas deferens and in guinea pig ileum Compound 24 was tested at the

single concentration of 100 nM against the effects of N/OFQ. In both preparations the

concentration response curves to N/OFQ obtained in the absence and presence of

Compound 24 were parallel and reached similar maximal effects. The estimated pKB

values were 8.28 ± 0.12 and 9.12 ± 0.19 in the rat vas deferens (Figure 34, left panel) and

guinea pig ileum (Figure 34, right panel), respectively. In these preparations, Compound

24 up to 1 µM was per se inactive.

-10 -9 -8 -7 -6 -50

20

40

60

80

100

ControlCompound 24 100 nM

Log[N/OFQ]

% C

ontro

l Tw

itch

-10 -9 -8 -7 -6 -560

70

80

90

100


Log[N/OFQ]

% C

ontr

ol T

witc

h

Rat Vas Deferens Guinea Pig Ileum

Figure 34. Concentration response curves to N/OFQ obtained in the absence (control) and presence of 100 nM Compound 24 in the electrically stimulated rat vas deferens (left panel) and guinea pig ileum (right panel). Data are mean ± s.e.m. of 4 separate experiments.

100

Finally, Compound 24 at 1 µM did not modify the inhibitory effects of DPDPE in

the mouse vas deferens (control: pEC50 (95% confidence limit) 8.38 (8.20 - 8.56), Emax, 98

± 1%; 1 µM Compound 24: pEC50 8.30 (7.80 - 8.80), Emax, 99 ± 1%) or those evoked by

dermorphin in the guinea pig ileum (control: pEC50 8.52 (8.35 - 8.69), Emax, 98 ± 3%; 1

µM Compound 24: pEC50, 8.51 (8.35 - 8.67), Emax, 95 ± 3%).

Tail withdrawal assay - In tail withdrawal experiments, mice injected with vehicle (either

i.c.v. or i.t.) displayed tail withdrawal latencies of approximately 5 s that were stable over

the time course of the experiment (Figure 35). In line with previous studies, N/OFQ (1

nmole) applied i.c.v. significantly reduced tail withdrawal latency with a maximal effect

(about 50% reduction in tail withdrawal latency) obtained at 5 min (AUC[0-15 min] vehicle 77

± 5; 1 nmole of N/OFQ 51 ± 3, p< 0.05). The i.p. administration of Compound 24 up to 10

mg/kg did not modify, per se, tail withdrawal latencies (AUC[0-15 min], 80 ± 7) but prevented

the pronociceptive effects of the natural peptide (AUC[0-15 min], 66 ± 5) (Figure 35, left

panel). When the same dose of N/OFQ was administered i.t., a statistically significant

antinociceptive effect was recorded (AUC[0-30 min] vehicle, 165 ± 14; 1 nmole of N/OFQ

346 ± 25, p < 0.05) (Figure 35, right panel). This antinociceptive effect was reduced by

Compound 24 (AUC[0-30 min], 226 ± 25) (Figure 35, right panel).

0 15 30 45 600

2

4

6

8

vehicleN/OFQ 1 nmolCompound 24 10 mg/kgN/OFQ + Compound 24

*

Time (min)

TW la

tenc

y (s

)

0 15 30 45 600

4

8

12

16

20

vehicleN/OFQ 1 nmolCompond 24 10 mg/kgN/OFQ + Compound 24

*

Time (min)

TW la

tenc

y (s

)

i.c.v. i.t.

Figure 35. Mouse tail withdrawal assay. Effects of Compound 24 (10 mg/kg i.p., 30 min pre-treatment) on the pronociceptive or antinociceptive effects induced by 1 nmole N/OFQ injected i.c.v. (left panel) or i.t. (right panel). Data are mean ± s.e.m. of 4 separate experiments. *p<0.05 versus vehicle according to ANOVA followed by the Dunnett's test.

101

Discussion

The present study extend previous findings (Goto et al., 2006) demonstrating that

Compound 24 binds with high affinity to the NOP receptor and behaves as a pure and

potent NOP receptor antagonist with high selectivity over classical opioid receptor. These

pharmacological features of Compound 24 were consistently observed in various assays

and preparations expressing the human recombinant as well as the animal native receptors.

In addition, the NOP antagonistic properties of Compound 24 have been confirmed in vivo

in mice in the tail withdrawal assay. Therefore, Compound 24 represents a valuable

research tool that should be included in the class of selective NOP receptor antagonists and

used in future target validation studies.

In receptor binding studies Compound 24 displayed very high affinity for the NOP

receptor. The pKi value calculated from the present experiments (9.62) is virtually

superimposable to that previously reported by Goto et al. (2006) (pIC50 9.57). These values

of affinity are similar to that reported for SB-612111 and higher than that of J-113397

(Ozaki et al., 2000; Spagnolo et al., 2007; Zaratin et al., 2004). In functional studies

performed on the human recombinant receptor ([35S]GTPγS binding and calcium

mobilization assays) and on animal native receptors from various species (mouse, rat,

guinea pig) Compound 24 consistently behaved as a pure NOP receptor antagonist

showing, in line with receptor binding studies, high values of potency (range 8.28 – 9.12).

The only result which was out of this range is that obtained in [35S]GTPγS binding studies

where Compound 24 displayed a pA2 of 9.98. However, this result, which is again

superimposable to that obtained by Goto et al. (2006) (pIC50 9.82), is expected on the basis

of what we have observed with several NOP receptor antagonists, including the peptides

[Nphe1]N/OFQ(1–13)-NH2 and UFP-101 (Calo et al., 2005; Calo et al., 2002a), and the

non-peptides J-113397, Trap-101, and SB-612111 (Spagnolo et al., 2007; Trapella et al.,

2006), that consistently showed, in this particular assay, values of potency approximately

10 fold higher than in the other tests. As previously suggested (Spagnolo et al., 2007), this

might be due to the higher receptor accessibility in membranes (where the [35S]GTPγS

binding assay is performed) than in whole cells or tissue preparations (where the other

assays are performed). Despite this minor difference, very similar antagonist potency

values were obtained in the different assays for Compound 24 demonstrating the

recombinant and native as well as species-specific NOP receptors are similarly sensitive to

this antagonist. This also applies to the NOP receptors expressed in rat periaqueductal gray

slices (Yan-Yu et al., 2008) and sympathetic neurons (Ruiz-Velasco et al., 2008) although

102

no quantitative data were reported in these latter studies. The consistency of Compound 24

potency values among preparations and species corroborates previous findings obtained

with peptide (Calo et al., 2000a; Calo et al., 2005; Calo et al., 2002a; Calo et al., 2002b)

and non-peptide (Bigoni et al., 2000a; Ozaki et al., 2000; Spagnolo et al., 2007; Trapella et

al., 2006; Zaratin et al., 2004) NOP antagonists and enable the following rank order of

antagonist potency to be proposed: Compound 24 (≈8.5) = SB-612111 (≈8.5) > J-113397

(≈8.0) > UFP-101 (≈7.5) = Trap-101 (≈7.5) > [Nphe1]N/OFQ(1–13)-NH2 (≈6.5).

With respect to the type of antagonism exerted by Compound 24, results obtained

in the [35S]GTPγS binding and mouse vas deferens assay by performing concentration

response curve to N/OFQ in the presence of increasing concentrations of antagonist are

clearly compatible with a competitive type of interaction between Compound 24 and

N/OFQ. Similar results were obtained by testing Compound 24 in the calcium mobilization

assay where a rightward displacement of the concentration response curve to N/OFQ was

recorded in response to increasing concentration of antagonist. However in these

experiments Compound 24 at the highest concentrations tested produced a slight but

statistically significant reduction of N/OFQ maximal effect. This apparent insurmountable

antagonism behaviour can be attributed to i) the transient nature of the calcium response

that may not allow equilibrium between agonist–antagonist competition to be reached thus

generating depression of the agonist response in the presence of high concentrations of

antagonist (Kenakin, 2004), ii) lack of stirring in the 96 well plate which is another source

of hemiequilibrium conditions (Kenakin, 2004), iii) a combination of the two factors. The

observation that a truly competitive antagonist produces a reduction of agonist maximal

effects in calcium mobilization assay is not uncommon. For instance, the urotensin-II

receptor antagonists urantide and UFP-803 competitively antagonized the contractile

effects of urotensin-II in the rat aorta bioassay while depressed maximal responses to the

agonist in calcium experiments performed on cells expressing the rat recombinant

urotensin-II receptor (Camarda et al., 2006). On this basis, we propose to classify

Compound 24 as a competitive NOP receptor antagonist.

Selectivity of action along with high affinity/potency and pure antagonist activity is

another important feature of a valuable research tool. This property of Compound 24 has

been evaluated in three sets of experiments over classical opioid receptors. In receptor

binding studies Compound 24 displayed approximately 1000 fold selectivity over classical

103

opioid receptors. A superimposable value of selectivity was found in functional studies

performed measuring calcium mobilization in cells expressing the Gαqi5 chimeric protein.

Finally at least 300 fold selectivity of Compound 24 for NOP over MOP and DOP was

obtained in bioassay experiments. These data confirm the impressive selectivity profile of

Compound 24 reported by Goto et al. (2006). Thus, in terms of selectivity over classical

opioid receptor Compound 24 seems to be similar to SB-612111 (Spagnolo et al., 2007;

Zaratin et al., 2004) and UFP-101 (Calo et al., 2002b) and certainly more selective then J-

113397 (Ozaki et al., 2000; Spagnolo et al., 2007) for which NOP independent effects

were reported in vivo (Koizumi et al., 2004). However more comprehensive selectivity

studies are needed against a large panel of receptors and ion channels to firmly classify

Compound 24 as a highly selective NOP receptor antagonist.

Collectively, these in vitro data confirm and extend previous findings (Goto et al.,

2006) demonstrating that Compound 24 is a pure, competitive, selective and potent NOP

receptor antagonist. This excellent in vitro pharmacological profile is associated with good

brain penetration after peripheral administration (Goto et al., 2006). Indeed, Compound 24

was reported to be active in vivo in mice where at 10 mg/kg it prevented the inhibitory

effects on locomotor activity of a non-peptide NOP agonist (Goto et al., 2006). Here we

assessed the in vivo effects of Compound 24 in the mouse tail withdrawal assay. N/OFQ

has been repeatedly demonstrated to exert in rodents pronociceptive and antinociceptive

effects following supraspinal and spinal injection, respectively (Zeilhofer et al., 2003). The

present results i.e. pronociceptive and antinociceptive response to 1 nmole N/OFQ given

i.c.v. and i.t., are therefore in line with the literature. Both of these in vivo actions of

N/OFQ are due to selective NOP receptor activation as consistently demonstrated by

knockout (Nazzaro et al., 2007; Nishi et al., 1997) and receptor antagonist (UFP-101 (Calo

et al., 2002b; Nazzaro et al., 2007), J-113397 (Ozaki et al., 2000; Ueda et al., 2000), SB-

612111 (Rizzi et al., 2007a; Zaratin et al., 2004)) studies. In line with these findings,

Compound 24 at 10 mg/kg antagonised both the pronociceptive and antinociceptive effects

evoked by N/OFQ when given spinally and supraspinally, respectively. This result

confirms on the one hand that Compound 24 is an effective antagonist at NOP receptors

regulating pain transmission in vivo and on the other that the effects of N/OFQ on pain

transmission are exclusively due to NOP receptor activation. Interestingly, Compound 24

at 10 mg/kg counteracted rather than fully prevented the effects of 1 nmole N/OFQ. Under

the same experimental conditions SB-612111 completely blocked the actions of the same

dose of N/OFQ at ten fold lower doses (i.e. 1 mg/kg) (Rizzi et al., 2007a). Therefore SB-

104

612111 appeared to be more potent than Compound 24 in vivo in the mouse tail

withdrawal assay. Since the in vitro potency of the two antagonists is similar (see above), it

can be proposed that in vivo potency differences may derive from better pharmacokinetic

properties of SB-612111 than Compound 24. However, this is merely speculation that

required rigorous experimental validation. Finally, the systemic administration of

Compound 24 at pharmacologically active doses did not modify per se tail withdrawal

latencies. Again this is in line with previous findings obtained with both receptor

antagonists (Ozaki et al., 2000; Rizzi et al., 2007a; Ueda et al., 2000; Zaratin et al., 2004)

and mice knockout for the NOP receptor gene (Nazzaro et al., 2007; Nishi et al., 1997),

and indicates that the endogenous N/OFQ-NOP receptor system is not activated by the

mild and acute stimulus employed for evoking the nociceptive response in this assay.

However, endogenous N/OFQergic signalling can be activated using more intense and

prolonged nociceptive stimuli such as formalin. In fact in the mouse formalin assay the

spinal antinociceptive action of endogenous N/OFQ seems to prevail over the supraspinal

pronociceptive effect as indicated by the pronociceptive phenotype of NOP knockout mice

(Depner et al., 2003; Rizzi et al., 2006) (recently confirmed in the acetic acid-induced

writhing test by Rizzi et al. (2008)) and by the pronociceptive effects elicited by NOP

antagonists e.g. systemic J-113397 (Rizzi et al., 2006).

In conclusion the present study demonstrated that Compound 24 is a pure,

competitive, selective and potent NOP receptor antagonist. The NOP antagonist properties

of Compound 24 were demonstrated in a large panel of in vitro assays and in vivo in the

mouse tail withdrawal assay. Based on its pharmacological profile, Compound 24 should

be included (together with J-113397, SB-612111 and UFP-101) in the list of potent and

selective NOP receptor antagonists to be tested in future target validation studies to firmly

define their therapeutic potential as innovative drugs for treating depression, Parkinson’s

disease and possibly sepsis.

105

3.4 Blending of chemical moieties of NOP receptor ligands: identification of a novel antagonist

Compound 24 displays some chemical characteristics typical of N/OFQ related

peptides (Figure 36), these include: i) a spacer of 12 atoms between the two phenyl rings

which corresponds to the Phe-Gly-Gly-Phe sequence of the N/OFQ message domain; ii) an

amide bond which is quite uncommon in non-peptide NOP ligands; iii) a N-benzyl amino

acid, a chemical moiety also present in the N-terminal part of the NOP receptor peptide

antagonists [Nphe1]N/OFQ(1-13)-NH2 (Guerrini et al., 2000a) and UFP-101 (Guerrini et

al., 2005). Based on these considerations, in the present study, the importance of the N-

benzyl D-Pro of Compound 24 was assessed by replacement with L- or D-Phe, and Nphe.

In addition, the amide bond of Compound 24 was substituted with other amide bond

isosters. Moreover, the Compound 24 spiroisobenzofurane nucleus was replaced with

chemical moieties derived from other non-peptide NOP ligands i.e. Ro 64-6198, SB-

612111 and J-113397 (Figure 36). The novel molecules were evaluated for their ability to

bind the human recombinant NOP receptor expressed in CHOhNOP cell membranes. The

molecule showing the highest affinity, i.e. Compound 35 has been further characterized at

the recombinant human NOP in the [35S]GTPγS binding and calcium mobilization assays

and at native NOP receptors expressed in isolated animal (mouse, rat, guinea-pig) tissues.

Figure 36. Chemical formulae of nociceptin/orphanin FQ receptor ligands.

H2NHN

NH

HN

O

O

O

O

N/OFQ message domain

NO

NH

ON

Compound 24

N

HO

N N

O

J-113397

N

Cl

Cl

OHSB-612111

NN

NHO

Ro 64-6198

106

Results and Discussion

Substitution into Compound 24 structure of the N-benzyl D-Pro with D or L-Phe

(compounds 9 and 10) or Nphe (compound 11) produced a profound (> 100 fold) loss of

NOP affinity suggesting a pivotal role of the N-benzyl D-Pro moiety for Compound 24

bioactivity (Table 8). This result is not surprising since the simple inversion of Pro

chirality was reported to generate an analog 200 fold less potent than Compound 24 (Goto

et al., 2006).

Modifications of the amide bond obtained by N-methylation (compound 12) or by

replacement with a methyleneamino (compound 13), an ester (compound 17), a

methyleneoxy (compound 20), or an alkene bond (compound 25), generated a drastic

reduction in NOP receptor binding or, in the case of compound 25, complete loss of

affinity. These results indicated that the amide bond represents a chemical feature crucial

for the bioactivity of Compound 24. It is worthy of note at this regard, that an amide bond

is a relatively uncommon chemical feature in non-peptide NOP ligands. However, this

chemical bond is also present in the NOP receptor antagonist JTC-801 and its chemical

modification (i.e. N-methylation and retro-inverso bond) was reported to be detrimental to

binding affinity (Shinkai et al., 2000). Thus, the amide bond might play a similarly

important role in both Compound 24 and JTC-801. In particular, according to the

pharmacophoric model for non-peptide NOP ligands proposed by Zaveri et al. (2005), the

amide bond may be part of the B-moiety that links and contributes to maintain in the

correct spatial disposition the A-moiety (important for affinity and selectivity) and the B-

moiety (important for efficacy).

Next, we considered replacement of the benzoisofurane in position 4 of the

Compound 24 piperidine scaffold with chemical moieties taken from the same position of

other high affinity NOP receptor ligands. The substitution in position 4 with 1-

phenylimidazolidin-4-one, the chemical group of the NOP receptor agonist Ro 64-6198

(compound 28), or with 1-ethylbenzoimidazol-2-one, the chemical group of the NOP

receptor antagonist J-113397 (compound 41), generated inactive molecules. Interestingly,

the insertion in position 4 of the piperidine scaffold of the 2,6-dichlorophenyl moiety

(Compound 35), a pharmacophore of the NOP receptor antagonist SB-612111, generated a

molecule with high affinity for the NOP receptor (pKi 9.14).

In particular Compound 35 is only 3 fold less potent than Compound 24. The

results obtained with this limited series of chimeric compounds suggest that it is possible to

combine pharmacophoric moieties taken from different NOP receptor ligands to generate

107

novel biologically active molecules. In particular, the chemical groups benzoisofurane and

2,6-dichlorophenyl seem to contribute to NOP receptor binding in a very similar manner.

This is corroborated by the finding that the substitution of D-Pro with L-Pro in the

chimeric NOP ligand compound 36 produced a 100-fold reduction in receptor affinity.

This result is in agreement with that previously observed with compounds 22 and 23 of the

Goto series (Goto et al., 2006). Thus, the D chirality of the Pro residue represents a crucial

requirement for both Compound 24 and Compound 35 for high affinity NOP receptor

recognition.

108

Table 8. Binding affinities at CHOhNOP cell membranes of Compound 24 and related compounds.

Compound Structure pKi

Compound 24 NN

H

N

O

O

9.62 ± 0.07

9 ON

HN

O

H2N

7.23 ± 0.19

10 ON

HN

O

H2N

7.23 ± 0.05

11 ONHN

O

NH

7.22 ± 0.08

12 NN

N

O

O

5.94 ± 0.28

13 NN

H

N

O

7.31 ± 0.06

17 NO

N

O

O

6.97 ± 0.02

20 NO

N

O

6.80 ± 0.80

25 N

N

O

< 5

28 NH

N

N

O

NH

O

N

< 5

35 Cl

Cl

NNH

O

N 9.14 ± 0.05

36 Cl

Cl

NNH

O

N 7.08 ± 0.02

41 NNH

N

O

N N

O

< 5

109

Based on its high affinity for the NOP receptor, Compound 35 was selected for

further pharmacological characterization. As shown in Figure 37 (left panel) in CHOhNOP

cell membranes N/OFQ concentration-dependently stimulated [35S]GTPγS binding with

pEC50 and Emax values of 8.91 and 10.93 ± 0.18 (stimulation factor), respectively. Over the

concentration range of 1 – 100 nM Compound 35 was inactive per se but produced a

rightward shift of the concentration response curve to N/OFQ in a parallel manner and

without modifying the maximal effect. Schild analysis of these data (Figure 37, right

panel) is compatible with a competitive type of antagonism with a pA2 value of 9.91. This

potency value is close to that previously reported for Compound 24 by Goto et al. (2006)

(pIC50 9.82) and by us (see Figure 31) (pA2 9.98).

-12 -11 -10 -9 -8 -7 -6 -5 -40

2

4

6

8

10

12

ControlCompound 35 1 nMCompound 35 10 nMCompound 35 100 nM

Log[N/OFQ]

Stim

ulat

ion

Fact

or

-9 -8 -7

0

1

2

3

pA2= 9.91r2= 0.91slope= 1.05 ± 0.10

Log[Compound 35]

Log(

CR

-1)

Figure 37. Concentration response curve to N/OFQ in the absence and presence of increasing concentrations of Compound 35 on [35S]GTPγS binding in CHOhNOP cell membranes. The corresponding Schild plot is shown in the right panel. Data are means ± s.e.m. of 4 separate experiments.

These results suggest that the benzoisofurane and 2,6-dichlorophenyl moieties of

Compound 24 and Compound 35 play a similar role not only in receptor binding but also

in determining pharmacological activity i.e. pure and competitive antagonism. This may

derive from the common ability of the spiro junction (Compound 24) and of the 2,6

dichloro substitution (Compound 35) to favor an orthogonal spatial disposition between

their piperidine and phenyl nuclei. Therefore, this conformational feature may likely be

crucial for both NOP receptor binding and antagonist activity.

110

The pharmacological actions of Compound 35 were further assessed at the hNOP

receptor coupled to calcium signalling via the chimeric protein Gαqi5; this assay has been

previously validated with a large panel of NOP receptor full and partial agonists and

antagonists (see section 3.1). In CHO cells stably expressing the hNOP receptor and the

Gαqi5 protein, N/OFQ produced a concentration-dependent stimulation of intracellular

calcium levels with a pEC50 of 9.24 and an Emax of 198 ± 12 % over the basal values.

Compound 35 was inactive per se up to 10 µM while inhibiting in a concentration-

dependent manner the stimulatory effect of 10 nM N/OFQ. A pKB value of 8.47 was

derived from these experiments (Table 9). This value of potency is approx 3 fold lower

than that obtained with Compound 24 (pKB 9.03, see Table 7).

Table 9. Compound 35 affinity/antagonist potency in various pharmacological assays.

CHOhNOP cell membranes

CHOhNOP /

Gαqi5 cells Electrically stimulated tissues

receptor binding

[35S]GTPγS binding

Ca2+ mobilization

mouse vas deferens

rat vas deferens

guinea pig ileum

pKi pA2 pKB pA2 pKB pKB

Compound 35 9.14 (9.04 – 9.24)

9.91 (9.23 – 10.59)

8.47 (8.31 – 8.63)

8.00 (7.32 – 8.68)

8.06 (7.58 – 8.54)

8.84 (8.64 – 9.04)

Data are means (CL95%) of at least 4 separate experiments.

The competitive antagonist behaviour of Compound 35 was confirmed at the

native NOP receptor expressed in the mouse vas deferens. In this preparation, N/OFQ

inhibited electrically evoked twitches in a concentration-dependent manner with pEC50 and

Emax values of 7.49 and -80 ± 2 %, respectively (Figure 38, left panel). Compound 35 was

inactive per se but caused a concentration-dependent (10 – 1000 nM) and parallel

rightward shift of the concentration response curve to N/OFQ without modifying the

agonist maximal effects. The relative Schild plot, depicted in Figure 38 (right panel),

demonstrated a competitive type of antagonism with a pA2 value of 8.00. This value of

potency is close to that previously reported for Compound 24 i.e. 8.44 (see Figure 33).

111

-10 -9 -8 -7 -6 -50

20

40

60

80

100

ControlCompound 35 10 nMCompound 35 100 nMCompound 35 1 µM

Log[N/OFQ]

% C

ontr

ol T

witc

h

-8 -7 -6

0

1

2

3

pA2= 8.00r2= 0.99slope= 1.06 ± 0.09

Log[Compound 35]

Log(

CR

-1)

Figure 38 Concentration response curve to N/OFQ in the absence and presence of increasing concentrations of Compound 35 on the electrically stimulated mouse vas deferens. The corresponding Schild plot is shown in the right panel. Data are means ± s.e.m. of 4 separate experiments.

Similar results were obtained in other N/OFQ sensitive preparations such as the

rat vas deferens and guinea pig ileum where Compound 35 at 100 nM antagonized N/OFQ

inhibitory action with pKb values of 8.06 and 8.84, respectively (Figure 39, Table 9).

Rat vas deferens

-10 -9 -8 -7 -6 -50

20

40

60

80

100


Log[N/OFQ]

% C

ontro

l Tw

itch

Guinea pig ileum

-10 -9 -8 -7 -6 -560

70

80

90

100


Log[N/OFQ]

% C

ontro

l Tw

itch

Figure 39. Concentration response-curve to N/OFQ obtained in the absence (control) and presence of Compound 35 (100nM) in the electrically stimulated rat vas deferens (left panel) and in guinea pig ileum (right panel). Data are means ± s.e.m of 3 separate experiments.

112

Finally the selectively of action of Compound 35 over classical opioid receptors

was assessed in animal tissues expressing native receptors and at recombinant human

proteins. Compound 35 at 1 µM was inactive per se and did not modify the inhibitory

effects elicited by the DOP selective agonist DPDPE in the mouse vas deferens (control

pEC50 8.38 (CL95% 8.20 – 8.56), Emax -98 ± 1%; 1 µM Compound 35 pEC50 8.44 (CL95%

8.26 – 8.62), Emax -98 ± 1%) or those produced by the MOP agonist dermorphin in the

guinea pig ileum (control pEC50 8.52 (CL95% 8.35 – 8.69), Emax -90 ± 3%; 1 µM

Compound 35 pEC50 8.44 (CL95% 8.19 – 8.69), Emax -85 ± 5%).

Results obtained with Compound 35 and the universal opioid receptor antagonist

naloxone in selectivity studies performed with receptor binding and calcium mobilization

assays are summarized in tables 10 and 11, respectively. Compound 35 up to 10 µM did

not displace [3H]DPN from DOP sites and showed very low affinity for MOP and KOP

receptors (Table 10). In contrast, naloxone did not bind the NOP receptor up to 10 µM,

while it displaced [3H]DPN from classical opioid receptors with very high affinity for

MOP and lower affinities for KOP and DOP (Table 10).

Table 10. Affinities of Compound 35 and naloxone at NOP and classical opioid receptors expressed in CHO cell membranes. receptor NOP MOP DOP KOP radioligand [3H]N/OFQ [3H]DPN [3H]DPN [3H]DPN naloxone < 6 9.25

(9.04 – 9.46)7.67

(7.59 – 7.75) 8.35

(8.20 – 8.50)

Compound 35 9.14 (9.04 – 9.24)

6.72 (6.47 – 6.97) < 6 6.50

(6.37 – 6.63) Data are mean (CL95%) of 4 separate experiments. Naloxone data on classical opioid receptors are from Vergura et al. (2008).

Similar results were found in functional studies performed measuring calcium

mobilization in CHO cells stably expressing the hNOP receptor or classical opioid

receptors and the Gαqi5 protein (Table 11). Dermorphin, DPDPE and dynorphin A were

used in these experiments as agonists for the MOP, DOP and KOP receptors, respectively.

All produced a concentration-dependent increase in calcium levels with the following

pEC50 values: 7.93 (CL95% 7.67 – 8.19), 8.82 (CL95% 8.43 – 9.21), 8.47 (CL95% 8.16 –

113

8.78). Naloxone inhibited the effects of these agonists showing higher potency at MOP

than KOP and DOP (Table 11), and being inactive against N/OFQ. Compound 35 was at

least 300 fold less potent at classical opioid receptors than at the NOP receptor (Table 11).

Table 11. Antagonist potencies of Compound 35 and naloxone evaluated in calcium mobilization experiments performed in CHO cells expressing NOP or classical opioid receptors and the Gαqi5 protein. receptor NOP MOP DOP KOP

agonist N/OFQ 10 nM

Dermorphin 100 nM

DPDPE 100 nM

Dynorphin A 100 nM

naloxone < 6 9.09

(8.73 – 9.45)7.32

(6.80 – 7.84) 7.14

(6.60 – 7.68)

Compound 35 8.47 (8.31 – 8.63)

6.11 (5.92 – 6.30) < 6 < 6

Data are mean (CL95%) of 4 separate experiments made in duplicate.

These pharmacological results confirmed that Compound 35 behaves as a pure,

potent and competitive NOP receptor antagonist. Moreover selectivity studies

demonstrated that the substitution of the benzoisofurane with the 2,6-dichlorophenyl

moiety not only allows maintenance of a high affinity and antagonist potency but also high

selectivity of action over classical opioid receptors.

3.5 General conclusions

The present thesis summarizes the work we performed in the last three years in the

field of N/OFQ and its receptor. Part of this work was performed during 2007 in Prof.

Lambert’s laboratories at University of Leicester as part of the Joint Ferrara-Leicester PhD

programme.

Following the formal identification of the receptor NOP and of its endogenous

ligand N/OFQ, an extensive search has started to assess the biological functions regulated

by this peptide-receptor system and to foresee the therapeutic indications of drugs

interacting selectively with the NOP receptor. In parallel, academic and industrial

laboratories generated new molecules that selectively activate or block the NOP receptor

thus providing the pharmacological tools needed for target validation studies.

114

The major aim of this work was detailed in vitro and in vivo pharmacological

characterization of novel ligands for the NOP receptor designed and synthesized by the

medicinal chemistry group of Prof. Salvadori. To this aim, we develop and use different in

vitro and in vivo assays: in vitro studies were performed measuring receptor and

[35S]GTPγS binding and calcium mobilization in cells expressing the recombinant NOP

receptor as well as using N/OFQ sensitive tissues from different species (mouse, rat,

guinea pig). In vivo studies were conducted using an analgesiometric assay such as the tail

withdrawal tests after both supraspinal and spinal administration. Moreover some in vitro

and in vivo experiments were performed using NOP(+/+) and NOP(-/-) mice for

investigating the involvement of the NOP receptor in the actions exerted by the novel

ligands.

Our major contributions to this field can be summarized below:

1) We confirmed that it is possible to use the Gαqi5 chimeric protein to force

classical opioid and NOP receptors to signal via the calcium pathway. [Ca2+]i

levels were monitored using the fluorometer FlexStation II. A panel of full and

partial agonists as well as antagonists were assessed in calcium mobilization

experiments demonstrating that that the FlexStation II – Gαqi5 NOP receptor

calcium assay represents a very useful strategy for the screening of NOP

receptor ligands particularly for antagonists. In fact, results obtained with this

class of ligands in the Gαqi5 NOP receptor calcium assay are superimposable to

those collected with more classical Gi based biochemical assays performed on

recombinant or native NOP receptors or with bioassays performed on isolated

tissues expressing native NOP receptors, with the exception of ligands

characterized by slow kinetics of interaction with the NOP receptor.

2) Parallel experiments, performed on mouse and rat tissues, confirmed previous

findings demonstrating that ZP120 behaves as a high potency NOP selective

partial agonist. In fact in both preparations ZP120 mimicked the effects of

N/OFQ showing higher potency but lower maximal effects. The effects of

N/OFQ and those elicited by ZP120 have been evaluated in the presence of two

NOP receptor antagonists: UFP-101 and J-113397. The antagonist sensitivity of

115

ZP120 effects (J-113397 sensitive and UFP-101 resistant) is not

superimposable to that of the endogenous NOP ligand N/OFQ (J-113397 and

UFP-101 sensitive). However the exclusive involvement of the NOP receptor

protein in the in vitro as well as in vivo effects of ZP120 (and N/OFQ) was

clearly demonstrated by knockout studies. Therefore the different sensitivity of

N/OFQ and ZP120 to UFP-101 might be attributed to their diverse binding and

activation of the NOP receptor. In vivo, ZP120 has been shown to mimic

N/OFQ actions displaying higher potency and longer lasting effects than the

natural ligand. These pharmacological features make ZP120 a promising and

innovative drug candidate for treating heart failure and for exploiting the

therapeutic benefits deriving from a prolonged partial activation of peripheral

NOP receptors.

3) Compound 24 has been recently identified as a novel non-peptide selective

antagonist (Goto et al., 2006). Our major goal was to perform an extensive in

vitro and in vivo pharmacological characterization of the actions of this

interesting molecule. Our findings derived from competition binding and

functional studies on CHOhNOP, calcium mobilization assay on CHOhNOP co-

expressing the Gαqi5 chimeric protein, bioassay studies on native receptors and

in vivo in the tail withdrawal assay, demonstrated that Compound 24 behaves as

one of the most potent and selective non-peptide NOP antagonists. It is worth

noting that synthesis of Compound 24 is relatively easy when compared to that

of other non-peptide NOP antagonists such as J-113397 and SB-612111.

4) A novel ligand has been identified from SAR studies of the NOP receptor

antagonist Compound 24, this compound is named Compound 35. Based on the

novel structure of Compound 24 the group of Prof. Salvadori synthesized

twelve new derivates focusing on the N-benzyl-D-proline, amide bond and

benzoisofurane moieties. This latter structure was substituted with moieties

taken from known non-peptide NOP ligands such as Ro 64-6198, SB-612111

and J-113397. The new derivates were evaluated for their ability to bind the

human recombinant NOP receptor and the molecule showing the highest

affinity (Compound 35) has been further characterized. In vitro studies were

performed measuring receptor and [35S]GTPγS binding and calcium

116

mobilization in cells expressing the recombinant NOP receptor as well as using

N/OFQ sensitive tissues. The pharmacological results confirmed that

Compound 35 behaves as a pure, potent and competitive NOP receptor

antagonist. Moreover selectivity studies demonstrated that the substitution of

the benzoisofurane with the 2,6-dichlorophenyl moiety not only allows

maintenance of a high affinity and antagonist potency but also high selectivity

of action over classical opioid receptors. Based on its pharmacological profile

Compound 35 might be useful in future in vivo studies aimed at investigating

the possible therapeutic indications of selective NOP antagonists.

The development of new NOP receptor antagonists has to be considered of primary

interest particularly in the field of pain (Fioravanti et al., 2008; Zeilhofer et al., 2003),

obesity (Przydzial et al., 2008), memory disorders (Jinsmaa et al., 2000), sepsis (Carvalho

et al., 2008; Williams et al., 2008) and mood (Gavioli et al., 2006). In addition, very recent

studies performed by the group of Prof. Morari (Marti et al., 2005; Marti et al., 2004a;

Marti et al., 2004b; Marti et al., 2008; Marti et al., 2007) provided very robust evidence

that NOP antagonists are worthy of development as innovative drugs for Parkinson’s

disease.

A useful comparison between peptide and non-peptide NOP receptor antagonists

is summarized in Table 12.

117

Table 12. Affinity and potency values of the peptide and non-peptide NOP receptor antagonists in different in vitro assays. Data are mean of at least 4 experiments.

CHOhNOP Electrically

Stimulated Tissues Cell membranes Whole cells

Receptor

Binding

[35S]GTPγS

Binding

Calcium

Assay mVD rVD gpI

pKi pA2 pKB pKB pKB pKB

UFP-101 10.24 9.12 7.66 7.29 7.30 7.18

J-113397 8.42 8.71 7.88 7.53 7.97 7.69

Trap-101 8.65 8.55 7.93 7.75 7.68 7.51

SB-612111 9.18 9.70 8.16 8.50 8.20 8.40

Compound 24 9.62 9.98 9.03 8.44 8.28 9.12

Compound 35 9.14 9.91 8.47 8.00 8.06 8.84

As shown in Table 12, all the NOP receptor antagonists were found to bind to the

NOP receptor with high affinity (in the nanomolar range). For each compound pA2 values

obtained in [35S]GTPγS binding experiments on CHOhNOP were higher comparing with all

the other tests. This might be due to the higher receptor availability in membranes (where

the [35S]GTPγS is performed) than in whole cells or tissues preparations (where the other

tests are performed). Despite this minor difference, very similar potency values were

obtained in different assays for all the antagonists. UFP-101, represents the most potent

NOP peptide antagonist. J-113397 has been largely used as a standard non-peptide NOP

antagonist. Among non-peptide molecules, SB-612111 is one of the most potent and

selective NOP receptor antagonist identified to date. In vitro, in most of the preparations,

Compound 24 has shown potency values higher than those obtained by SB-612111.

However, the in vivo potency of Compound 24 seems to be lower than that of SB-612111.

Moreover, synthesis of Compound 24 and Compound 35 is easier compared with that of

both J-113397 and SB-612111. Collectively these data suggest the following order of

potency of antagonists: Compound 24 = Compound 35 = SB-612111 > J-113397 > UFP-

101 = Trap-101. Based on the findings summarized in this thesis Compound 24 and

Compound 35 should be included (together with J-113397, SB-612111 and UFP-101) in

the list of potent and selective NOP receptor antagonists to be used in future target

118

validation studies for firmly defining the therapeutic potential of selective NOP antagonists

as innovative drugs.

119

Bibliography

ADAM, G., CESURA, A., GALLEY, G., JENCK, F., MONSMA, F., ROVER, S. & WICHMANN, J. (1998). New 8-substitute-1,3,8-triazaspiro[4.5]decan-4-on derivatives - useful for the treatment of diseases related to the Orphanin FQ receptor e.g. psychiatric, neurological and physiological disorders. Switzerland: Hoffmann-La Roche.

AMANO, T., MATSUBAYASHI, H., TAMURA, Y. & TAKAHASHI, T. (2000). Orphanin FQ-induced outward current in rat hippocampus. Brain Res, 853, 269-74.

AMBO, A., HAMAZAKI, N., YAMADA, Y., NAKATA, E. & SASAKI, Y. (2001). Structure-activity studies on nociceptin analogues: ORL1 receptor binding and biological activity of cyclic disulfide-containing analogues of nociceptin peptides. J Med Chem, 44, 4015-8.

ANDERBERG, U.M., LIU, Z., BERGLUND, L. & NYBERG, F. (1998). Plasma levels on nociceptin in female fibromyalgia syndrome patients. Z Rheumatol, 57 Suppl 2, 77-80.

ARDUIN, M., SPAGNOLO, B., CALO, G., GUERRINI, R., CARRA, G., FISCHETTI, C., TRAPELLA, C., MARZOLA, E., MCDONALD, J., LAMBERT, D.G., REGOLI, D. & SALVADORI, S. (2007). Synthesis and biological activity of nociceptin/orphanin FQ analogues substituted in position 7 or 11 with Calpha,alpha-dialkylated amino acids. Bioorg Med Chem, 15, 4434-4443.

ARMSTEAD, W.M. (1999). Nociceptin/orphanin FQ dilates pial arteries by K(ATP) and K(ca) channel activation. Brain Res, 835, 315-23.

ARNDT, M.L., WU, D., SOONG, Y. & SZETO, H.H. (1999). Nociceptin/orphanin FQ increases blood pressure and heart rate via sympathetic activation in sheep. Peptides, 20, 465-70.

BASSO, M., RISSE, P.A., NALINE, E., CALO, G., GUERRINI, R., REGOLI, D. & ADVENIER, C. (2005). Nociceptin/orphanin FQ inhibits electrically induced contractions of the human bronchus via NOP receptor activation. Peptides, 26, 1492-1496.

BECKER, J.A., WALLACE, A., GARZON, A., INGALLINELLA, P., BIANCHI, E., CORTESE, R., SIMONIN, F., KIEFFER, B.L. & PESSI, A. (1999). Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library. J Biol Chem, 274, 27513-22.

BERGER, H., ALBRECHT, E., WALLUKAT, G. & BIENERT, M. (1999). Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ. Br J Pharmacol, 126, 555-8.

BERGER, H., BIGONI, R., ALBRECHT, E., RICHTER, R.M., KRAUSE, E., BIENERT, M. & CALO, G. (2000a). The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties. Peptides, 21, 1131-9.

BERGER, H., CALO, G., ALBRECHT, E., GUERRINI, R. & BIENERT, M. (2000b). [Nphe(1)]NC(1-13)NH(2) selectively antagonizes nociceptin/orphanin FQ-stimulated G-protein activation in rat brain. J Pharmacol Exp Ther, 294, 428-33.

BERTHELE, A., PLATZER, S., DWORZAK, D., SCHADRACK, J., MAHAL, B., BUTTNER, A., ASSMUS, H.P., WURSTER, K., ZIEGLGANSBERGER, W., CONRAD, B. & TOLLE, T.R. (2003). [3H]-nociceptin ligand-binding and nociceptin opioid receptor mrna expression in the human brain. Neuroscience, 121, 629-40.

BERTORELLI, R., CORRADINI, L., RAFIQ, K., TUPPER, J., CALO, G. & ONGINI, E. (1999). Nociceptin and the ORL-1 ligand [Phe1psi (CH2-NH)Gly2]nociceptin(1-13)NH2 exert anti-opioid effects in the Freund's adjuvant-induced arthritic rat model of chronic pain. Br J Pharmacol, 128, 1252-8.

BERZETEI-GURSKE, I.P., SCHWARTZ, R.W. & TOLL, L. (1996). Determination of activity for nociceptin in the mouse vas deferens. Eur J Pharmacol, 302, R1-2.

120

BES, B. & MEUNIER, J.C. (2003). Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH2. Biochem Biophys Res Commun, 310, 992-1001.

BIGNAN, G.C., CONNOLLY, P.J. & MIDDLETON, S.A. (2005). Recent advances towards the discovery of ORL-1 receptor agonists and antagonists. Expert Opin Ther Patents, 15, 357-388.

BIGONI, R., CALO, G., RIZZI, A., GUERRINI, R., DE RISI, C., HASHIMOTO, Y., HASHIBA, E., LAMBERT, D.G. & REGOLI, D. (2000a). In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist. Naunyn Schmiedebergs Arch Pharmacol, 361, 565-8.

BIGONI, R., GIULIANI, S., CALO, G., RIZZI, A., GUERRINI, R., SALVADORI, S., REGOLI, D. & MAGGI, C.A. (1999). Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies. Naunyn Schmiedebergs Arch Pharmacol, 359, 160-7.

BIGONI, R., RIZZI, A., RIZZI, D., BECKER, J.A., KIEFFER, B.L., SIMONIN, F., REGOLI, D. & CALO, G. (2000b). In vitro pharmacological profile of peptide III-BTD: a novel ligand for nociceptin/orphanin FQ and opioid receptors. Life Sci, 68, 233-9.

BIGONI, R., RIZZI, D., RIZZI, A., CAMARDA, V., GUERRINI, R., LAMBERT, D.G., HASHIBA, E., BERGER, H., SALVADORI, S., REGOLI, D. & CALO, G. (2002). Pharmacological characterisation of [(pX)Phe4]nociceptin(1-13)amide analogues. 1. In vitro studies. Naunyn Schmiedebergs Arch Pharmacol, 365, 442-9.

BLAKLEY, G.G., POHORECKY, L.A. & BENJAMIN, D. (2004). Behavioral and endocrine changes following antisense oligonucleotide-induced reduction in the rat NOP receptor. Psychopharmacology (Berl), 171, 421-8.

BOLSER, D.C., MCLEOD, R.L., TULSHIAN, D.B. & HEY, J.A. (2001). Antitussive action of nociceptin in the cat. Eur J Pharmacol, 430, 107-11.

BOOM, A., MOLLEREAU, C., MEUNIER, J.C., VASSART, G., PARMENTIER, M., VANDERHAEGHEN, J.J. & SCHIFFMANN, S.N. (1999). Distribution of the nociceptin and nocistatin precursor transcript in the mouse central nervous system. Neuroscience, 91, 991-1007.

BREGOLA, G., ZUCCHINI, S., FRIGATI, L., CANDELETTI, S., ROMUALDI, P., REINSCHEID, R. & SIMONATO, M. (2002). Involvement of the neuropeptide orphanin FQ/nociceptin in kainate and kindling seizures and epileptogenesis. Epilepsia, 43 Suppl 5, 18-9.

BROCCARDO, M., AGOSTINI, S., PETRELLA, C., GUERRINI, R. & IMPROTA, G. (2008). Central and peripheral role of the nociceptin/orphaninFQ system on normal and disturbed colonic motor function and faecal pellet output in the rat. Neurogastroenterol Motil., 20, 939-48.

BROCCARDO, M., GUERRINI, R., PETRELLA, C. & IMPROTA, G. (2004). Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats. Peptides, 25, 1013-20.

BROOKES, Z.L., STEDMAN, E.N., GUERRINI, R., LAWTON, B.K., CALO, G. & LAMBERT, D.G. (2007). Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine. Am J Physiol Heart Circ Physiol., 293, H2977-85.

BROOKS, H., ELTON, C.D., SMART, D., ROWBOTHAM, D.J., MCKNIGHT, A.T. & LAMBERT, D.G. (1998). Identification of nociceptin in human cerebrospinal fluid: comparison of levels in pain and non-pain states. Pain, 78, 71-3.

BROWN, J.M., GOUTY, S., IYER, V., ROSENBERGER, J. & COX, B.M. (2006). Differential protection against MPTP or methamphetamine toxicity in dopamine neurons by deletion of ppN/OFQ expression. J Neurochem., 98, 495-505.

BUNZOW, J.R., SAEZ, C., MORTRUD, M., BOUVIER, C., WILLIAMS, J.T., LOW, M. & GRANDY, D.K. (1994). Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opioid receptor type. FEBS Lett., 347, 284-8.

121

BURNSIDE, J.L., RODRIGUEZ, L. & TOLL, L. (2000). Species differences in the efficacy of compounds at the nociceptin receptor (ORL1). Peptides, 21, 1147-54.

BUTOUR, J.L., MOISAND, C., MAZARGUIL, H., MOLLEREAU, C. & MEUNIER, J.C. (1997). Recognition and activation of the opioid receptor-like ORL 1 receptor by nociceptin, nociceptin analogs and opioids. Eur J Pharmacol, 321, 97-103.

BUTOUR, J.L., MOISAND, C., MOLLEREAU, C. & MEUNIER, J.C. (1998). [Phe1psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 is an agonist of the nociceptin (ORL1) receptor. Eur J Pharmacol, 349, R5-6.

CALO, G., GUERRINI, R., BIGONI, R., RIZZI, A., BIANCHI, C., REGOLI, D. & SALVADORI, S. (1998a). Structure-activity study of the nociceptin(1-13)-NH2 N-terminal tetrapeptide and discovery of a nociceptin receptor antagonist. J Med Chem, 41, 3360-6.

CALO, G., GUERRINI, R., BIGONI, R., RIZZI, A., MARZOLA, G., OKAWA, H., BIANCHI, C., LAMBERT, D.G., SALVADORI, S. & REGOLI, D. (2000a). Characterization of [Nphe(1)]nociceptin(1-13)NH(2), a new selective nociceptin receptor antagonist. Br J Pharmacol, 129, 1183-93.

CALO, G., GUERRINI, R., RIZZI, A., SALVADORI, S., BURMEISTER, M., KAPUSTA, D.R., LAMBERT, D.G. & REGOLI, D. (2005). UFP-101, a Peptide Antagonist Selective for the Nociceptin/Orphanin FQ Receptor. CNS Drug Rev, 11, 97-112.

CALO, G., GUERRINI, R., RIZZI, A., SALVADORI, S. & REGOLI, D. (2000b). Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol, 129, 1261-83.

CALO, G., RIZZI, A., BIGONI, R., GUERRINI, R., SALVADORI, S. & REGOLI, D. (2002a). Pharmacological profile of nociceptin/orphanin FQ receptors. Clin Exp Pharmacol Physiol., 29, 223-8.

CALO, G., RIZZI, A., BODIN, M., NEUGEBAUER, W., SALVADORI, S., GUERRINI, R., BIANCHI, C. & REGOLI, D. (1997). Pharmacological characterization of nociceptin receptor: an in vitro study. Can J Physiol Pharmacol, 75, 713-8.

CALO, G., RIZZI, A., BOGONI, G., NEUGEBAUER, V., SALVADORI, S., GUERRINI, R., BIANCHI, C. & REGOLI, D. (1996). The mouse vas deferens: a pharmacological preparation sensitive to nociceptin. Eur J Pharmacol, 311, R3-5.

CALO, G., RIZZI, A., MARZOLA, G., GUERRINI, R., SALVADORI, S., BEANI, L., REGOLI, D. & BIANCHI, C. (1998b). Pharmacological characterization of the nociceptin receptor mediating hyperalgesia in the mouse tail withdrawal assay. Br J Pharmacol, 125, 373-8.

CALO, G., RIZZI, A., RIZZI, D., BIGONI, R., GUERRINI, R., MARZOLA, G., MARTI, M., MCDONALD, J., MORARI, M., LAMBERT, D.G., SALVADORI, S. & REGOLI, D. (2002b). [Nphe1,Arg14,Lys15]nociceptin-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor. Br J Pharmacol, 136, 303-11.

CAMARDA, V., FISCHETTI, C., ANZELLOTTI, N., MOLINARI, P., AMBROSIO, C., KOSTENIS, E., REGOLI, D., TRAPELLA, C., GUERRINI, R., SALVADORI, S., CALO, G. (2009). Pharmacological profile of NOP receptors coupled with calcium signalling via the chimeric protein Gqi5. Naunyn-Schmiedeberg Archives of Pharmacology in press.

CAMARDA, V., SPAGNOL, M., SONG, W., VERGURA, R., ROTH, A.L., THOMPSON, J.P., ROWBOTHAM, D.J., GUERRINI, R., MARZOLA, E., SALVADORI, S., CAVANNI, P., REGOLI, D., DOUGLAS, S.A., LAMBERT, D.G. & CALO, G. (2006). In vitro and in vivo pharmacological characterization of the novel UT receptor ligand [Pen(5),DTrp(7),Dab(8)]urotensin II(4-11) (UFP-803). Br J Pharmacol, 147, 92-100.

CANDELETTI, S. & FERRI, S. (2000a). Effects of an antisense oligonucleotide to pronociceptin and long-term prevention of morphine actions by nociceptin. Peptides., 21, 1119-24.

CANDELETTI, S., GUERRINI, R., CALO, G., ROMUALDI, P. & FERRI, S. (2000b). Supraspinal and spinal effects of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 on nociception in the rat. Life Sci, 66, 257-64.

122

CARPENTER, K.J. & DICKENSON, A.H. (1998). Evidence that [Phe1 psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2, a peripheral ORL-1 receptor antagonist, acts as an agonist in the rat spinal cord. Br J Pharmacol, 125, 949-51.

CARRA, G., CALO, G., SPAGNOLO, B., GUERRINI, R., ARDUIN, M., MARZOLA, E., TRAPELLA, C., REGOLI, D. & SALVADORI, S. (2005a). Tryptophan replacement in the nociceptin/orphanin FQ receptor ligand Ac-RYYRWK-NH2. J Peptide Res, 66, 39-47.

CARRA, G., RIZZI, A., GUERRINI, R., BARNES, T.A., MCDONALD, J., HEBBES, C.P., MELA, F., KENIGS, V.A., MARZOLA, G., RIZZI, D., GAVIOLI, E., ZUCCHINI, S., REGOLI, D., MORARI, M., SALVADORI, S., ROWBOTHAM, D.J., LAMBERT, D.G., KAPUSTA, D.R. & CALO, G. (2005b). [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor. J Pharmacol Exp Ther, 312, 1114-23.

CARVALHO, D., PETRONILHO, F., VUOLO, F., MACHADO, R.A., CONSTANTINO, L., GUERRINI, R., CALO, G., GAVIOLI, E.C., STRECK, E.L. & DAL-PIZZOL, F. (2008). The nociceptin/orphanin FQ-NOP receptor antagonist effects on an animal model of sepsis. Intensive Care Med, 34, 2284-90.

CHAMPION, H.C., CZAPLA, M.A. & KADOWITZ, P.J. (1997). Nociceptin, an endogenous ligand for the ORL1 receptor, decreases cardiac output and total peripheral resistance in the rat. Peptides, 18, 729-32.

CHAMPION, H.C., PIERCE, R.L. & KADOWITZ, P.J. (1998). Nociceptin, a novel endogenous ligand for the ORL1 receptor, dilates isolated resistance arteries from the rat. Regul Pept, 78, 69-74.

CHAN, J.S., YUNG, L.Y., LEE, J.W., WU, Y.L., PEI, G. & WONG, Y.H. (1998). Pertussis toxin-insensitive signaling of the ORL1 receptor: coupling to Gz and G16 proteins. J Neurochem., 71, 2203-10.

CHEN, Y., FAN, Y., LIU, J., MESTEK, A., TIAN, M., KOZAK, C.A. & YU, L. (1994). Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family. FEBS Lett., 347, 279-83.

CHEN, Y., MESTEK, A., LIU, J., HURLEY, J.A. & YU, L. (1993). Molecular cloning and functional expression of a mu-opioid receptor from rat brain. Mol Pharmacol, 44, 8-12.

CHEN, Y.L., LI, A.H., YEH, T.H., CHOU, A.H. & WANG, H.L. (2009). Nocistatin and nociceptin exert opposite effects on the excitability of central amygdala nucleus-periaqueductal gray projection neurons. Mol Cell Neurosci., 40, 76-88.

CHENG, Y. & PRUSOFF, W.H. (1973). Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol., 22, 3099-108.

CHIOU, L.C. & FAN, S.H. (2002). CompB (J-113397), selectively and competitively antagonizes nociceptin activation of inwardly rectifying K(+) channels in rat periaqueductal gray slices. Neuropharmacology, 42, 987-92.

CHIOU, L.C., LIAO, Y.Y., FAN, P.C., KUO, P.H., WANG, C.H., RIEMER, C. & PRINSSEN, E.P. (2007). Nociceptin/orphanin FQ peptide receptors: pharmacology and clinical implications. Curr Drug Targets., 8, 117-35.

CHU, X., XU, N., LI, P., MAO, L. & WANG, J.Q. (1999). Inhibition of cardiovascular activity following microinjection of novel opioid-like neuropeptide nociceptin (orphanin FQ) into the rat rostral ventrolateral medulla. Brain Res, 829, 134-42.

CICCOCIOPPO, R., BIONDINI, M., ANTONELLI, L., WICHMANN, J., JENCK, F. & MASSI, M. (2002). Reversal of stress- and CRF-induced anorexia in rats by the synthetic nociceptin/orphanin FQ receptor agonist, Ro 64-6198. Psychopharmacology (Berl), 161, 113-9.

CICCOCIOPPO, R., CIPPITELLI, A., ECONOMIDOU, D., FEDELI, A. & MASSI, M. (2004). Nociceptin/orphanin FQ acts as a functional antagonist of corticotropin-releasing factor to inhibit its anorectic effect. Physiol Behav, 82, 63-8.

123

CICCOCIOPPO, R., ECONOMIDOU, D., RIMONDINI, R., SOMMER, W., MASSI, M. & HEILIG, M. (2007). Buprenorphine reduces alcohol drinking through activation of the nociceptin/orphanin FQ-NOP receptor system. Biol Psychiatry., 61, 4-12.

CICCOCIOPPO, R., MARTIN-FARDON, R., WEISS, F. & MASSI, M. (2001). Nociceptin/orphanin FQ inhibits stress- and CRF-induced anorexia in rats. Neuroreport, 12, 1145-9.

CICCOCIOPPO, R., PANOCKA, I., POLIDORI, C., REGOLI, D. & MASSI, M. (1999). Effect of nociceptin on alcohol intake in alcohol-preferring rats. Psychopharmacology (Berl), 141, 220-4.

CIVELLI, O. (2005). GPCR deorphanizations: the novel, the known and the unexpected transmitters. Trends Pharmacol Sci, 26, 15-9.

CIVELLI, O. (2008). The orphanin FQ/nociceptin (OFQ/N) system. Results Probl Cell Differ, 46, 1-25.

CONKLIN, B.R., FARFEL, Z., LUSTIG, K.D., JULIUS, D. & BOURNE, H.R. (1993). Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature., 363, 274-6.

CONNOR, M., VAUGHAN, C.W., CHIENG, B. & CHRISTIE, M.J. (1996a). Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro. Br J Pharmacol, 119, 1614-8.

CONNOR, M., YEO, A. & HENDERSON, G. (1996b). The effect of nociceptin on Ca2+ channel current and intracellular Ca2+ in the SH-SY5Y human neuroblastoma cell line. Br J Pharmacol, 118, 205-7.

CORBOZ, M.R., FERNANDEZ, X., EGAN, R.W. & HEY, J.A. (2001). Inhibitory activity of nociceptin/orphanin FQ on capsaicin-induced bronchoconstriction in the guinea-pig. Life Sci, 69, 1203-11.

COWARD, P., CHAN, S.D., WADA, H.G., HUMPHRIES, G.M. & CONKLIN, B.R. (1999). Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal Biochem, 270, 242-8.

COX, B.M., CHAVKIN, C., CHRISTIE, M.J., CIVELLI, O., EVANS, C., HAMON, M.D., HOELLT, V., KIEFFER, B., KITCHEN, I., MCKNIGHT, A.T., MEUNIER, J.C. & PORTOGHESE, P.S. (2000). Opioid receptors. In The IUPHAR Compendium of Receptor Characterization and Classification. ed Girdlestone, D. pp. 321-333. London: IUPHAR Media Ltd.

CURRO, D., YOO, J.H., ANDERSON, M., SONG, I., DEL VALLE, J. & OWYANG, C. (2001). Molecular cloning of the orphanin FQ receptor gene and differential tissue expression of splice variants in rat. Gene, 266, 139-45.

DANIELSON, P.B., HOVERSTEN, M.T., FITZPATRICK, M., SCHRECK, C., AKIL, H. & DORES, R.M. (2001). Sturgeon orphanin, a molecular "fossil" that bridges the gap between the opioids and orphanin FQ/nociceptin. J Biol Chem, 276, 22114-9.

DARLISON, M.G., GRETEN, F.R., HARVEY, R.J., KREIENKAMP, H.J., STUHMER, T., ZWIERS, H., LEDERIS, K. & RICHTER, D. (1997). Opioid receptors from a lower vertebrate (Catostomus commersoni): sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution. Proc Natl Acad Sci U S A., 94, 8214-9.

DAUTZENBERG, F.M., WICHMANN, J., HIGELIN, J., PY-LANG, G., KRATZEISEN, C., MALHERBE, P., KILPATRICK, G.J. & JENCK, F. (2001). Pharmacological characterization of the novel nonpeptide orphanin FQ/nociceptin receptor agonist Ro 64-6198: rapid and reversible desensitization of the ORL1 receptor in vitro and lack of tolerance in vivo. J Pharmacol Exp Ther, 298, 812-9.

DE RISI, C., PIERO POLLINI, G., TRAPELLA, C., PERETTO, I., RONZONI, S. & GIARDINA, G.A. (2001). A new synthetic approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dih ydro-benzimidazol-2-one(J-113397), the first non-peptide ORL-1 receptor antagonist. Bioorg Med Chem., 9, 1871-7.

124

DEPNER, U.B., REINSCHEID, R.K., TAKESHIMA, H., BRUNE, K. & ZEILHOFER, H.U. (2003). Normal sensitivity to acute pain, but increased inflammatory hyperalgesia in mice lacking the nociceptin precursor polypeptide or the nociceptin receptor. Eur J Neurosci, 17, 2381-7.

DEVINE, D.P., REINSCHEID, R.K., MONSMA, F.J., JR., CIVELLI, O. & AKIL, H. (1996). The novel neuropeptide orphanin FQ fails to produce conditioned place preference or aversion. Brain Res, 727, 225-9.

DHAWAN, B.N., CESSELIN, F., RAGHUBIR, R., REISINE, T., BRADLEY, P.B., PORTOGHESE, P.S. & HAMON, M. (1996). International Union of Pharmacology. XII. Classification of opioid receptors. Pharmacol Rev., 48, 567-92.

DI GIANNUARIO, A., PIERETTI, S., CATALANI, A. & LOIZZO, A. (1999). Orphanin FQ reduces morphine-induced dopamine release in the nucleus accumbens: a microdialysis study in rats. Neurosci Lett., 272, 183-6.

DI GIANNUARIO, A., RIZZI, A., PIERETTI, S., GUERRINI, R., BERTORELLI, R., SALVADORI, S., REGOLI, D. & CALO, G. (2001). Studies on the antinociceptive effect of [Nphe1]nociceptin(1-13)NH2 in mice. Neurosci Lett, 316, 25-8.

DOOLEY, C.T. & HOUGHTEN, R.A. (1996). Orphanin FQ: receptor binding and analog structure activity relationships in rat brain. Life Sci, 59, PL23-9.

DOOLEY, C.T., SPAETH, C.G., BERZETEI-GURSKE, I.P., CRAYMER, K., ADAPA, I.D., BRANDT, S.R., HOUGHTEN, R.A. & TOLL, L. (1997). Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1. J Pharmacol Exp Ther, 283, 735-41.

DREWS, J. (2000). Drug discovery: a historical perspective. Science., 287, 1960-4. ECONOMIDOU, D., FEDELI, A., FARDON, R.M., WEISS, F., MASSI, M. & CICCOCIOPPO, R. (2006a).

Effect of novel nociceptin/orphanin FQ-NOP receptor ligands on ethanol drinking in alcohol-preferring msP rats. Peptides., 27, 3299-306.

ECONOMIDOU, D., POLICANI, F., ANGELLOTTI, T., MASSI, M., TERADA, T. & CICCOCIOPPO, R. (2006b). Effect of novel NOP receptor ligands on food intake in rats. Peptides., 27, 775-83.

ERB, K., LIEBEL, J.T., TEGEDER, I., ZEILHOFER, H.U., BRUNE, K. & GEISSLINGER, G. (1997). Spinally delivered nociceptin/orphanin FQ reduces flinching behaviour in the rat formalin test. Neuroreport, 8, 1967-70.

ERSPAMER, V., MELCHIORRI, P., ERSPAMER, C.F. & NEGRI, L. (1978). Polypeptides of the amphibian skin active on the gut and their mammalian counterparts. Adv Exp Med Biol, 106, 51-64.

ERTSEY, C., HANTOS, M., BOZSIK, G. & TEKES, K. (2004). Circulating nociceptin levels during the cluster headache period. Cephalalgia, 24, 280-3.

ERTSEY, C., HANTOS, M., BOZSIK, G. & TEKES, K. (2005). Plasma nociceptin levels are reduced in migraine without aura. Cephalalgia., 25, 261-6.

EVANS, C.J., KEITH, D.E., JR., MORRISON, H., MAGENDZO, K. & EDWARDS, R.H. (1992). Cloning of a delta opioid receptor by functional expression. Science, 258, 1952-1955.

FANTIN, M., FISCHETTI, C., TRAPELLA, C. & MORARI, M. (2007). Nocistatin inhibits 5-hydroxytryptamine release in the mouse neocortex via presynaptic Gi/o protein linked pathways. Br J Pharmacol., 152, 549-55.

FERNANDEZ, F., MISILMERI, M.A., FELGER, J.C. & DEVINE, D.P. (2004). Nociceptin/orphanin FQ increases anxiety-related behavior and circulating levels of corticosterone during neophobic tests of anxiety. Neuropsychopharmacology, 29, 59-71.

FIORAVANTI, B. & VANDERAH, T.W. (2008). The ORL-1 receptor system: are there opportunities for antagonists in pain therapy? Curr Top Med Chem., 8, 1442-51.

FISCHER, A., FORSSMANN, W.G. & UNDEM, B.J. (1998). Nociceptin-induced inhibition of tachykinergic neurotransmission in guinea pig bronchus. J Pharmacol Exp Ther, 285, 902-7.

125

FISET, M.E., GILBERT, C., POUBELLE, P.E. & POULIOT, M. (2003). Human neutrophils as a source of nociceptin: a novel link between pain and inflammation. Biochemistry, 42, 10498-505.

FLORIN, S., SUAUDEAU, C., MEUNIER, J.C. & COSTENTIN, J. (1996). Nociceptin stimulates locomotion and exploratory behaviour in mice. Eur J Pharmacol, 317, 9-13.

FLORIN, S., SUAUDEAU, C., MEUNIER, J.C. & COSTENTIN, J. (1997). Orphan neuropeptide NocII, a putative pronociceptin maturation product, stimulates locomotion in mice. Neuroreport, 8, 705-7.

FUKUDA, K., KATO, S., MORI, K., NISHI, M. & TAKESHIMA, H. (1993). Primary structures and expression from cDNAs of rat opioid receptor delta- and mu-subtypes. FEBS Lett., 327, 311-4.

FUKUDA, K., KATO, S., MORI, K., NISHI, M., TAKESHIMA, H., IWABE, N., MIYATA, T., HOUTANI, T. & SUGIMOTO, T. (1994). cDNA cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett., 343, 42-6.

GAVIOLI, E.C. & CALO, G. (2006). Antidepressant- and anxiolytic-like effects of nociceptin/orphanin FQ receptor ligands. Naunyn Schmiedebergs Arch Pharmacol, 372, 319-30.

GAVIOLI, E.C., DUARTE, F.S., GUERRINI, R., CALO, G., RAE, G.A. & TC, M.D.L. (2008). GABA(A) signalling is involved in N/OFQ anxiolytic-like effects but not in nocistatin anxiogenic-like action as evaluated in the mouse elevated plus maze. Peptides., 29, 1404-12.

GAVIOLI, E.C., MARZOLA, G., GUERRINI, R., BERTORELLI, R., ZUCCHINI, S., DE LIMA, T.C., RAE, G.A., SALVADORI, S., REGOLI, D. & CALO, G. (2003). Blockade of nociceptin/orphanin FQ-NOP receptor signalling produces antidepressant-like effects: pharmacological and genetic evidences from the mouse forced swimming test. Eur J Neurosci, 17, 1987-90.

GAVIOLI, E.C., RAE, G.A., CALO, G., GUERRINI, R. & DE LIMA, T.C. (2002). Central injections of nocistatin or its C-terminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plus-maze test. Br J Pharmacol, 136, 764-72.

GAVIOLI, E.C., RIZZI, A., MARZOLA, G., ZUCCHINI, S., REGOLI, D. & CALO, G. (2007). Altered anxiety-related behavior in nociceptin/orphanin FQ receptor gene knockout mice. Peptides, 27, 1229-39.

GAVIOLI, E.C., VAUGHAN, C.W., MARZOLA, G., GUERRINI, R., MITCHELL, V.A., ZUCCHINI, S., DE LIMA, T.C., RAE, G.A., SALVADORI, S., REGOLI, D. & CALO, G. (2004). Antidepressant-like effects of the nociceptin/orphanin FQ receptor antagonist UFP-101: new evidence from rats and mice. Naunyn Schmiedebergs Arch Pharmacol, 369, 547-553.

GIULIANI, S., LECCI, A. & MAGGI, C.A. (2000). Nociceptin and neurotransmitter release in the periphery. Peptides, 21, 977-84.

GIULIANI, S., LECCI, A., TRAMONTANA, M. & MAGGI, C.A. (1998). The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats. Br J Pharmacol, 124, 1566-72.

GIULIANI, S. & MAGGI, C.A. (1996). Inhibition of tachykinin release from peripheral endings of sensory nerves by nociceptin, a novel opioid peptide. Br J Pharmacol, 118, 1567-9.

GIULIANI, S. & MAGGI, C.A. (1997a). Prejunctional modulation by nociceptin of nerve-mediated inotropic responses in guinea-pig left atrium. Eur J Pharmacol, 332, 231-6.

GIULIANI, S., TRAMONTANA, M., LECCI, A. & MAGGI, C.A. (1997b). Effect of nociceptin on heart rate and blood pressure in anaesthetized rats. Eur J Pharmacol, 333, 177-9.

GOELDNER, C., REISS, D., WICHMANN, J., MEZIANE, H., KIEFFER, B.L. & OUAGAZZAL, A.M. (2008). Nociceptin receptor impairs recognition memory via interaction with NMDA receptor-dependent mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in the hippocampus. J Neurosci., 28, 2190-8.

GONZALEZ-NUNEZ, V., GONZALEZ-SARMIENTO, R. & RODRIGUEZ, R.E. (2003). Cloning and characterization of a full-length pronociceptin in zebrafish: evidence of the existence of

126

two different nociceptin sequences in the same precursor. Biochim Biophys Acta, 1629, 114-8.

GOTO, Y., ARAI-OTSUKI, S., TACHIBANA, Y., ICHIKAWA, D., OZAKI, S., TAKAHASHI, H., IWASAWA, Y., OKAMOTO, O., OKUDA, S., OHTA, H. & SAGARA, T. (2006). Identification of a novel spiropiperidine opioid receptor-like 1 antagonist class by a focused library approach featuring 3D-pharmacophore similarity. J Med Chem., 49, 847-9.

GRIEBEL, G., PERRAULT, G. & SANGER, D.J. (1999). Orphanin FQ, a novel neuropeptide with anti-stress-like activity. Brain Res, 836, 221-4.

GRISEL, J.E., FARRIER, D.E., WILSON, S.G. & MOGIL, J.S. (1998). [Phe1psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 acts as an agonist of the orphanin FQ/nociceptin receptor in vivo. Eur J Pharmacol, 357, R1-3.

GRISEL, J.E., MOGIL, J.S., BELKNAP, J.K. & GRANDY, D.K. (1996). Orphanin FQ acts as a supraspinal, but not a spinal, anti-opioid peptide. Neuroreport, 7, 2125-9.

GU, H., HU, D., HONG, X.R., MAO, J., CUI, Y., HUI, N. & SHA, J.Y. (2003). [Changes and significance of orphanin and serotonin in patients with postpartum depression]. Zhonghua Fu Chan Ke Za Zhi, 38, 727-8.

GUERRINI, R., CALO, G., BIGONI, R., RIZZI, A., VARANI, K., TOTH, G., GESSI, S., HASHIBA, E., HASHIMOTO, Y., LAMBERT, D.G., BOREA, P.A., TOMATIS, R., SALVADORI, S. & REGOLI, D. (2000a). Further studies on nociceptin-related peptides: discovery of a new chemical template with antagonist activity on the nociceptin receptor. J Med Chem, 43, 2805-13.

GUERRINI, R., CALO, G., BIGONI, R., RIZZI, D., RIZZI, A., ZUCCHINI, M., VARANI, K., HASHIBA, E., LAMBERT, D.G., TOTH, G., BOREA, P.A., SALVADORI, S. & REGOLI, D. (2001). Structure-activity studies of the Phe(4) residue of nociceptin(1-13)-NH(2): identification of highly potent agonists of the nociceptin/orphanin FQ receptor. J Med Chem, 44, 3956-64.

GUERRINI, R., CALO, G., LAMBERT, D.G., CARRA, G., ARDUIN, M., BARNES, T.A., MCDONALD, J., RIZZI, D., TRAPELLA, C., MARZOLA, E., ROWBOTHAM, D.J., REGOLI, D. & SALVADORI, S. (2005). N- and C-terminal modifications of nociceptin/orphanin FQ generate highly potent NOP receptor ligands. J Med Chem, 48, 1421-7.

GUERRINI, R., CALO, G., RIZZI, A., BIANCHI, C., LAZARUS, L.H., SALVADORI, S., TEMUSSI, P.A. & REGOLI, D. (1997). Address and message sequences for the nociceptin receptor: a structure-activity study of nociceptin-(1-13)-peptide amide. J Med Chem, 40, 1789-93.

GUERRINI, R., CALO, G., RIZZI, A., BIGONI, R., BIANCHI, C., SALVADORI, S. & REGOLI, D. (1998). A new selective antagonist of the nociceptin receptor. Br J Pharmacol, 123, 163-5.

GUERRINI, R., CALO, G., RIZZI, A., BIGONI, R., RIZZI, D., REGOLI, D. & SALVADORI, S. (2000b). Structure-activity relationships of nociceptin and related peptides: comparison with dynorphin A. Peptides., 21, 923-33.

GUERRINI, R., CARRA, G., CALO, G., TRAPELLA, C., MARZOLA, E., RIZZI, D., REGOLI, D. & SALVADORI, S. (2004). Nonpeptide/peptide chimeric ligands for the nociceptin/orphanin FQ receptor: design, synthesis and in vitro pharmacological activity. J Pept Res, 63, 477-84.

GUERRINI, R., RIZZI, D., ZUCCHINI, M., TOMATIS, R., REGOLI, D., CALO, G. & SALVADORI, S. (2003). Nociceptin/Orphanin FQ(1-13)NH2 analogues modified in the Phe1-Gly2 peptide bond. Bioorg Med Chem Lett, 13, 365-8.

GUMUSEL, B., HAO, Q., HYMAN, A., CHANG, J.K., KAPUSTA, D.R. & LIPPTON, H. (1997). Nociceptin: an endogenous agonist for central opioid like1 (ORL1) receptors possesses systemic vasorelaxant properties. Life Sci, 60, PL141-5.

GUNDUZ, O., RIZZI, A., BALDISSEROTTO, A., GUERRINI, R., SPAGNOLO, B., GAVIOLI, E.C., KOCSIS, L., MAGYAR, A., BENYHE, S., BORSODI, A. & CALO, G. (2006). In vitro and in vivo pharmacological characterization of the nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-ol. Eur J Pharmacol., 539, 39-48.

127

HALFORD, W.P., GEBHARDT, B.M. & CARR, D.J. (1995). Functional role and sequence analysis of a lymphocyte orphan opioid receptor. J Neuroimmunol., 59, 91-101.

HANTOS, M.B., SZALAY, F., LAKATOS, P.L., HEGEDUS, D., FIRNEISZ, G., REICZIGEL, J., TOROK, T. & TEKES, K. (2002). Elevated plasma nociceptin level in patients with Wilson disease. Brain Res Bull, 58, 311-3.

HAO, J.X., WIESENFELD-HALLIN, Z. & XU, X.J. (1997). Lack of cross-tolerance between the antinociceptive effect of intrathecal orphanin FQ and morphine in the rat. Neurosci Lett, 223, 49-52.

HAO, J.X., XU, I.S., WIESENFELD-HALLIN, Z. & XU, X.J. (1998). Anti-hyperalgesic and anti-allodynic effects of intrathecal nociceptin/orphanin FQ in rats after spinal cord injury, peripheral nerve injury and inflammation. Pain, 76, 385-93.

HARA, N., MINAMI, T., OKUDA-ASHITAKA, E., SUGIMOTO, T., SAKAI, M., ONAKA, M., MORI, H., IMANISHI, T., SHINGU, K. & ITO, S. (1997). Characterization of nociceptin hyperalgesia and allodynia in conscious mice. Br J Pharmacol, 121, 401-8.

HASHIBA, E., HARRISON, C., GALO, G., GUERRINI, R., ROWBOTHAM, D.J., SMITH, G. & LAMBERT, D.G. (2001). Characterisation and comparison of novel ligands for the nociceptin/orphanin FQ receptor. Naunyn Schmiedebergs Arch Pharmacol, 363, 28-33.

HASHIBA, E., LAMBERT, D.G., FARKAS, J., TOTH, G. & SMITH, G. (2002a). Comparison of the binding of [(3)H]nociceptin/orphaninFQ(1-13)NH(2), [(3)H]nociceptin/orphaninFQ(1-17)OH and [(125)I]Tyr(14)nociceptin/orphaninFQ(1-17)OH to recombinant human and native rat cerebrocortical nociceptin/orphanin FQ receptors. Neurosci Lett, 328, 5-8.

HASHIBA, E., LAMBERT, D.G., JENCK, F., WICHMANN, J. & SMITH, G. (2002b). Characterisation of the non-peptide nociceptin receptor agonist, Ro64-6198 in Chinese hamster ovary cells expressing recombinant human nociceptin receptors. Life Sci, 70, 1719-25.

HASHIMOTO, Y., CALO, G., GUERRINI, R., SMITH, G. & LAMBERT, D.G. (2000). Antagonistic effects of [Nphe1]nociceptin(1-13)NH2 on nociceptin receptor mediated inhibition of cAMP formation in Chinese hamster ovary cells stably expressing the recombinant human nociceptin receptor. Neurosci Lett, 278, 109-12.

HAWES, B.E., GRAZIANO, M.P. & LAMBERT, D.G. (2000). Cellular actions of nociceptin: transduction mechanisms. Peptides, 21, 961-7.

HAWKINSON, J.E., ACOSTA-BURRUEL, M. & ESPITIA, S.A. (2000). Opioid activity profiles indicate similarities between the nociceptin/orphanin FQ and opioid receptors. Eur J Pharmacol, 389, 107-14.

HAYASHI, S., HIRAO, A., IMAI, A., NAKAMURA, H., MURATA, Y., OHASHI, K. & NAKATA, E. (2009). Novel Non-Peptide Nociceptin/Orphanin FQ Receptor Agonist, 1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole: Design, Synthesis, and Structure-Activity Relationship of Oral Receptor Occupancy in the Brain for Orally Potent Antianxiety Drug (1, 2). J Med Chem, 6, 6.

HIRAMATSU, M. & INOUE, K. (1999a). Effects of nocistatin on nociceptin-induced impairment of learning and memory in mice. Eur J Pharmacol, 367, 151-5.

HIRAMATSU, M. & INOUE, K. (1999b). Nociceptin/orphanin FQ and nocistatin on learning and memory impairment induced by scopolamine in mice. Br J Pharmacol, 127, 655-60.

HIRAO, A., IMAI, A., SUGIE, Y., TAMURA, T., SHIMOKAWA, H. & TOIDE, K. (2008a). Pharmacological properties of a novel nociceptin/orphanin FQ receptor agonist, 2-(3,5-dimethylpiperazin-1-yl)-1-[1-(1-methylcyclooctyl)piperidin-4-yl]-1H-benzim idazole, with anxiolytic potential. Eur J Pharmacol., 579, 189-95.

HIRAO, A., IMAI, A., SUGIE, Y., YAMADA, Y., HAYASHI, S. & TOIDE, K. (2008b). Pharmacological characterization of the newly synthesized nociceptin/orphanin FQ-receptor agonist 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole as an anxiolytic agent. J Pharmacol Sci., 106, 361-8.

128

HO, M., CORBETT, A.D. & MCKNIGHT, A.T. (2000). Characterization of the ORL(1) receptor on adrenergic nerves in the rat anococcygeus muscle. Br J Pharmacol, 131, 349-55.

HORVATH, A., FOLHOFFER, A., LAKATOS, P.L., HALOSZ, J., ILLYES, G., SCHAFF, Z., HANTOS, M.B., TEKES, K. & SZALAY, F. (2004). Rising plasma nociceptin level during development of HCC: a case report. World J Gastroenterol, 10, 152-4.

HOUTANI, T., NISHI, M., TAKESHIMA, H., SATO, K., SAKUMA, S., KAKIMOTO, S., UEYAMA, T., NODA, T. & SUGIMOTO, T. (2000). Distribution of nociceptin/orphanin FQ precursor protein and receptor in brain and spinal cord: a study using in situ hybridization and X-gal histochemistry in receptor-deficient mice. J Comp Neurol, 424, 489-508.

HUGHES, J., KOSTERLITZ, H.W. & LESLIE, F.M. (1975). Effect of morphine on adrenergic transmission in the mouse vas deferens. Assessment of agonist and antogonist potencies of narcotic analgesics. Br J Pharmacol., 53, 371-81.

HYLDEN, J.L. & WILCOX, G.L. (1980). Intrathecal morphine in mice: a new technique. Eur J Pharmacol, 17, 313-316.

IKEDA, K., WATANABE, M., ICHIKAWA, T., KOBAYASHI, T., YANO, R. & KUMANISHI, T. (1998). Distribution of prepro-nociceptin/orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems. J Comp Neurol, 399, 139-51.

INOUE, M., SHIMOHIRA, I., YOSHIDA, A., ZIMMER, A., TAKESHIMA, H., SAKURADA, T. & UEDA, H. (1999). Dose-related opposite modulation by nociceptin/orphanin FQ of substance P nociception in the nociceptors and spinal cord. J Pharmacol Exp Ther, 291, 308-13.

ISHIAMA, K., TEREDA, T., OYAMA, T. & OHGI, T. (2001). Peptide derivatives and medicinal compositions.

ISHIHARA, S., MINOWA, S., TSUCHIYA, S., HORIE, S., WATANABE, K. & MURAYAMA, T. (2002). Gastric acid secretion stimulated by centrally injected nociceptin in urethane-anesthetized rats. Eur J Pharmacol, 441, 105-14.

JENCK, F., MOREAU, J.L., MARTIN, J.R., KILPATRICK, G.J., REINSCHEID, R.K., MONSMA, F.J., JR., NOTHACKER, H.P. & CIVELLI, O. (1997). Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natl Acad Sci U S A, 94, 14854-8.

JENCK, F., WICHMANN, J., DAUTZENBERG, F.M., MOREAU, J.L., OUAGAZZAL, A.M., MARTIN, J.R., LUNDSTROM, K., CESURA, A.M., POLI, S.M., ROEVER, S., KOLCZEWSKI, S., ADAM, G. & KILPATRICK, G. (2000). A synthetic agonist at the orphanin FQ/nociceptin receptor ORL1: anxiolytic profile in the rat. Proc Natl Acad Sci U S A, 97, 4938-43.

JIA, Y., WANG, X., APONTE, S.I., RIVELLI, M.A., YANG, R., RIZZO, C.A., CORBOZ, M.R., PRIESTLEY, T. & HEY, J.A. (2002). Nociceptin/orphanin FQ inhibits capsaicin-induced guinea-pig airway contraction through an inward-rectifier potassium channel. Br J Pharmacol, 135, 764-70.

JINSMAA, Y., TAKAHASHI, M., FUKUNAGA, H. & YOSHIKAWA, M. (2000). Retro-nociceptin methylester, a peptide with analgesic and memory-enhancing activity. Life Sci, 67, 3095-101.

JUDD, A., B., FEIN, J., TO, T., LI, X., SILBERSTEIN, L. & EVANS, C.J. (1996). Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol., 368, 229-51.

JUDD, A.K., KAUSHANSKAYA, A., TUTTLE, D.J., SANCHEZ, A., KHROYAN, T., POLGAR, W. & TOLL, L. (2003). N-terminal modifications leading to peptide ORL1 partial agonists and antagonists. J Pept Res, 62, 191-8.

JUDD, A.K., TUTTLE, D.J., JONES, R.W., SANCHEZ, A., POLGAR, W., BERZETEI-GURSKE, I. & TOLL, L. (2004). Structure-activity studies on high affinity NOP-active hexapeptides. J Pept Res, 64, 87-94.

KAMEI, J., MATSUNAWA, Y., MIYATA, S., TANAKA, S. & SAITOH, A. (2004). Effects of nociceptin on the exploratory behavior of mice in the hole-board test. Eur J Pharmacol, 489, 77-87.

129

KAMEI, J., OHSAWA, M., KASHIWAZAKI, T. & NAGASE, H. (1999). Antinociceptive effects of the ORL1 receptor agonist nociceptin/orphanin FQ in diabetic mice. Eur J Pharmacol, 370, 109-16.

KAPUSTA, D.R. (2000). Neurohumoral effects of orphanin FQ/nociceptin: relevance to cardiovascular and renal function. Peptides, 21, 1081-99.

KAPUSTA, D.R., BURMEISTER, M.A., CALO, G., GUERRINI, R., GOTTLIEB, H.B. & KENIGS, V.A. (2005a). Functional Selectivity of Nociceptin/Orphanin FQ Peptide Receptor Partial Agonists on Cardiovascular and Renal Function. J Pharmacol Exp Ther, 314, 643-51.

KAPUSTA, D.R., CHANG, J.K. & KENIGS, V.A. (1999a). Central administration of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)-NH2 and orphanin FQ/nociceptin (OFQ/N) produce similar cardiovascular and renal responses in conscious rats. J Pharmacol Exp Ther, 289, 173-80.

KAPUSTA, D.R., DAYAN, L.A. & KENIGS, V.A. (2002). Nociceptin/orphanin FQ modulates the cardiovascular, but not renal, responses to stress in spontaneously hypertensive rats. Clin Exp Pharmacol Physiol., 29, 254-9.

KAPUSTA, D.R. & KENIGS, V.A. (1999b). Cardiovascular and renal responses produced by central orphanin FQ/nociceptin occur independent of renal nerves. Am J Physiol, 277, R987-95.

KAPUSTA, D.R., SEZEN, S.F., CHANG, J.K., LIPPTON, H. & KENIGS, V.A. (1997). Diuretic and antinatriuretic responses produced by the endogenous opioid-like peptide, nociceptin (orphanin FQ). Life Sci, 60, PL15-21.

KAPUSTA, D.R., THORKILDSEN, C., KENIGS, V.A., MEIER, E., VINGE, M.M., QUIST, C. & PETERSEN, J.S. (2005b). Pharmacodynamic Characterization of ZP120 (Ac-RYYRWKKKKKKK-NH2), a Novel, Functionally Selective Nociceptin/Orphanin FQ Peptide Receptor Partial Agonist with Sodium-Potassium-Sparing Aquaretic Activity. J Pharmacol Exp Ther, 314, 652-60.

KAWAMOTO, H., OZAKI, S., ITOH, Y., MIYAJI, M., ARAI, S., NAKASHIMA, H., KATO, T., OHTA, H. & IWASAWA, Y. (1999). Discovery of the first potent and selective small molecule opioid receptor-like (ORL1) antagonist: 1-[(3R,4R)-1-cyclooctylmethyl-3- hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one (J-113397). J Med Chem., 42, 5061-3.

KENAKIN, T. (2004). A Pharmacology Primer. San Diego: Elsevier Academic Press. KEST, B., HOPKINS, E., PALMESE, C.A., CHEN, Z.P., MOGIL, J.S. & PINTAR, J.E. (2001). Morphine

tolerance and dependence in nociceptin/orphanin FQ transgenic knock-out mice. Neuroscience, 104, 217-22.

KHO, S.T., LOPEZ, I.A., EVANS, C., ISHIYAMA, A. & ISHIYAMA, G. (2006). Immunolocalization of orphanin FQ in rat cochlea. Brain Res., 1113, 146-52.

KIEFFER, B.L., BEFORT, K., GAVERIAUX-RUFF, C. & HIRTH, C.G. (1992). The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc Natl Acad Sci U S A, 89, 12048-12052.

KING, M., CHANG, A. & PASTERNAK, G.W. (1998). Functional blockade of opioid analgesia by orphanin FQ/nociceptin. Biochem Pharmacol, 55, 1537-40.

KING, M.A., ROSSI, G.C., CHANG, A.H., WILLIAMS, L. & PASTERNAK, G.W. (1997). Spinal analgesic activity of orphanin FQ/nociceptin and its fragments. Neurosci Lett, 223, 113-6.

KITAYAMA, M., BARNES, T.A., CARRA, G., MCDONALD, J., CALO, G., GUERRINI, R., ROWBOTHAM, D.J., SMITH, G. & LAMBERT, D.G. (2003). Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys10,14]N/OFQ(1-14)NH2 and c[Nphe1,Cys10,14]N/OFQ(1-14)NH2. Naunyn Schmiedebergs Arch Pharmacol, 368, 528-37.

KNOFLACH, F., REINSCHEID, R.K., CIVELLI, O. & KEMP, J.A. (1996). Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. J Neurosci, 16, 6657-64.

130

KO, M.C., NAUGHTON, N.N., TRAYNOR, J.R., SONG, M.S., WOODS, J.H., RICE, K.C. & MCKNIGHT, A.T. (2002a). Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors. Br J Pharmacol, 135, 943-50.

KO, M.C., WEI, H., WOODS, J.H. & KENNEDY, R.T. (2006). Effects of Intrathecally Administered Nociceptin/Orphanin FQ in Monkeys: Behavioral and Mass Spectrometric Studies. J Pharmacol Exp Ther., 318, 1257-64.

KO, M.H., KIM, Y.H., WOO, R.S. & KIM, K.W. (2002b). Quantitative analysis of nociceptin in blood of patients with acute and chronic pain. Neuroreport, 13, 1631-3.

KOBAYASHI, T., IKEDA, K., TOGASHI, S., ITOH, N. & KUMANISHI, T. (1997). Effects of sigma ligands on the nociceptin/orphanin FQ receptor co-expressed with the G-protein-activated K+ channel in Xenopus oocytes. Br J Pharmacol, 120, 986-7.

KOCSIS, L., OROSZ, G., MAGYAR, A., AL-KHRASANI, M., KATO, E., RONAI, A.Z., BES, B., MEUNIER, J.C., GUNDUZ, O., TOTH, G., BORSODI, A. & BENYHE, S. (2004). Nociceptin antagonism: probing the receptor by N-acetyl oligopeptides. Regul Pept, 122, 199-207.

KOIZUMI, M., SAKOORI, K., MIDORIKAWA, N. & MURPHY, N.P. (2004). The NOP (ORL1) receptor antagonist Compound B stimulates mesolimbic dopamine release and is rewarding in mice by a non-NOP-receptor-mediated mechanism. Br J Pharmacol, 143, 53-62.

KOSTENIS, E., WAELBROECK, M. & MILLIGAN, G. (2005). Techniques: promiscuous Galpha proteins in basic research and drug discovery. Trends Pharmacol Sci., 26, 595-602.

KOSTER, A., MONTKOWSKI, A., SCHULZ, S., STUBE, E.M., KNAUDT, K., JENCK, F., MOREAU, J.L., NOTHACKER, H.P., CIVELLI, O. & REINSCHEID, R.K. (1999). Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice. Proc Natl Acad Sci U S A, 96, 10444-9.

KOTLINSKA, J., WICHMANN, J., RAFALSKI, P., TALAREK, S., DYLAG, T. & SILBERRING, J. (2003). Non-peptidergic OP4 receptor agonist inhibits morphine antinociception but does not influence morphine dependence. Neuroreport, 14, 601-4.

KROWICKI, Z.K. & KAPUSTA, D.R. (2006). Tonic nociceptinergic inputs to neurons in the hypothalamic paraventricular nucleus contribute to sympathetic vasomotor tone and water and electrolyte homeostasis in conscious rats. J Pharmacol Exp Ther., 317, 446-53.

KUZMIN, A., SANDIN, J., TERENIUS, L. & OGREN, S.O. (2003). Acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in mice: effects of opioid receptor-like 1 receptor agonists and naloxone. J Pharmacol Exp Ther, 304, 310-8.

LACHOWICZ, J.E., SHEN, Y., MONSMA, F.J., JR. & SIBLEY, D.R. (1995). Molecular cloning of a novel G protein-coupled receptor related to the opiate receptor family. J Neurochem., 64, 34-40.

LAI, C.C., WU, S.Y., CHEN, C.T. & DUN, N.J. (2000). Nociceptin inhibits rat sympathetic preganglionic neurons in situ and in vitro. Am J Physiol Regul Integr Comp Physiol, 278, R592-7.

LAMBERT, D.G. (2008). The nociceptin/orphanin FQ receptor: a target with broad therapeutic potential. Nat Rev Drug Discov., 7, 694-710.

LARSEN, B.D. (1999). Pharmacologically active peptide conjugates having reduced tendency towards enzymatic hydrolysis.

LARSEN, B.D., PETERSEN, J.S., HARLOW, K. & KAPUSTA, D.R. (2001). Novel peptide conjugates. LAUDENBACH, V., CALO, G., GUERRINI, R., LAMBOLEY, G., BENOIST, J.F., EVRARD, P. &

GRESSENS, P. (2001). Nociceptin/orphanin FQ exacerbates excitotoxic white-matter lesions in the murine neonatal brain. J Clin Invest, 107, 457-66.

LAURSEN, S.E. & BELKNAP, J.K. (1986). Intracerebroventricular injections in mice. Some methodological refinements. J Pharmacol Methods, 16, 355-7.

LAZZERI, M., CALO', G., SPINELLI, M., MALAGUTI, S., GUERRINI, R., SALVADORI, S., BENEFORTI, P., REGOLI, D. & TURINI, D. (2006). Daily intravesical instillation of 1 mg

131

nociceptin/orphanin FQ for the control of neurogenic detrusor overactivity - a multicenter, placebo controlled, randomized exploratory study. J Urol, 176, 2098-2102.

LAZZERI, M., CALO, G., SPINELLI, M., GUERRINI, R., BENEFORTI, P., SANDRI, S., ZANOLLO, A., REGOLI, D. & TURINI, D. (2001). Urodynamic and clinical evidence of acute inhibitory effects of intravesical nociceptin/orphanin FQ on detrusor overactivity in humans: a pilot study. J Urol, 166, 2237-40.

LAZZERI, M., CALO, G., SPINELLI, M., GUERRINI, R., SALVADORI, S., BENEFORTI, P., SANDRI, S., REGOLI, D. & TURINI, D. (2003). Urodynamic effects of intravesical nociceptin/orphanin FQ in neurogenic detrusor overactivity: a randomized, placebo-controlled, double-blind study. Urology, 61, 946-50.

LE MAITRE, E., DAUBEUF, F., DUTERTE-BOUCHER, D., COSTENTIN, J. & LEROUX-NICOLLET, I. (2006). Coupling of ORL1 (NOP) receptor to G proteins is decreased in the nucleus accumbens of anxious relative to non-anxious mice. Brain Res., 1110, 144-9.

LE PEN, G., WICHMANN, J., MOREAU, J.L. & JENCK, F. (2002). The orphanin receptor agonist RO 64-6198 does not induce place conditioning in rats. Neuroreport, 13, 451-4.

LECCI, A., GIULIANI, S., MEINI, S. & MAGGI, C.A. (2000). Nociceptin and the micturition reflex. Peptides, 21, 1007-21.

LEE, K., NICHOLSON, J.R. & MCKNIGHT, A.T. (1997). Nociceptin hyperpolarises neurones in the rat ventromedial hypothalamus. Neurosci Lett, 239, 37-40.

LEMAIRE, S., MAGNAN, J. & REGOLI, D. (1978). Rat vas deferens: a specific bioassay for endogenous opioid peptides. Br J Pharmacol., 64, 327-9.

LETCHWORTH, S.R., MATHIS, J.P., ROSSI, G.C., BODNAR, R.J. & PASTERNAK, G.W. (2000). Autoradiographic localization of (125)I[Tyr(14)]orphanin FQ/nociceptin and (125)I[Tyr(10)]orphanin FQ/nociceptin(1-11) binding sites in rat brain. J Comp Neurol, 423, 319-29.

LEVENTHAL, L., MATHIS, J.P., ROSSI, G.C., PASTERNAK, G.W. & BODNAR, R.J. (1998). Orphan opioid receptor antisense probes block orphanin FQ-induced hyperphagia. Eur J Pharmacol, 349, R1-3.

LI, J., ISOZAKI, K., OKADA, K., MATSUSHIMA, A., NOSE, T., COSTA, T. & SHIMOHIGASHI, Y. (2008). Designed modification of partial agonist of ORL1 nociceptin receptor for conversion into highly potent antagonist. Bioorg Med Chem., 16, 2635-44.

LI, X., KEITH, D.E., JR. & EVANS, C.J. (1996). Multiple opioid receptor-like genes are identified in diverse vertebrate phyla. FEBS Lett., 397, 25-9.

LOWRY, O.H., ROSENBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem, 193, 265-275.

LUO, C., KUMAMOTO, E., FURUE, H., CHEN, J. & YOSHIMURA, M. (2002). Nociceptin inhibits excitatory but not inhibitory transmission to substantia gelatinosa neurones of adult rat spinal cord. Neuroscience, 109, 349-58.

LUTFY, K., EITAN, S., BRYANT, C.D., YANG, Y.C., SALIMINEJAD, N., WALWYN, W., KIEFFER, B.L., TAKESHIMA, H., CARROLL, F.I., MAIDMENT, N.T. & EVANS, C.J. (2003). Buprenorphine-induced antinociception is mediated by mu-opioid receptors and compromised by concomitant activation of opioid receptor-like receptors. J Neurosci, 23, 10331-7.

LUTFY, K., KHALIQ, I., CARROLL, F.I. & MAIDMENT, N.T. (2002). Orphanin FQ/nociceptin blocks cocaine-induced behavioral sensitization in rats. Psychopharmacology (Berl), 164, 168-76.

LUTFY, K., SHARZA, S.A. & MAIDMENT, N.T. (1999). Tolerance develops to the inhibitory effect of orphanin FQ on morphine-induced antinociception in the rat. Neuroreport, 10, 103-6.

MADAMBA, S.G., SCHWEITZER, P. & SIGGINS, G.R. (1999). Nociceptin augments K(+) currents in hippocampal CA1 neurons by both ORL-1 and opiate receptor mechanisms. J Neurophysiol, 82, 1776-85.

132

MADEDDU, P., SALIS, M.B., MILIA, A.F., EMANUELI, C., GUERRINI, R., REGOLI, D. & CALO, G. (1999). Cardiovascular effects of nociceptin in unanesthetized mice. Hypertension, 33, 914-9.

MAMIYA, T., NODA, Y., NISHI, M., TAKESHIMA, H. & NABESHIMA, T. (1998). Enhancement of spatial attention in nociceptin/orphanin FQ receptor-knockout mice. Brain Res, 783, 236-40.

MAMIYA, T., NODA, Y., NISHI, M., TAKESHIMA, H. & NABESHIMA, T. (1999). Nociceptin system plays a role in the memory retention: involvement of naloxone benzoylhydrazone binding sites. Neuroreport., 10, 1171-5.

MANABE, T., NODA, Y., MAMIYA, T., KATAGIRI, H., HOUTANI, T., NISHI, M., NODA, T., TAKAHASHI, T., SUGIMOTO, T., NABESHIMA, T. & TAKESHIMA, H. (1998). Facilitation of long-term potentiation and memory in mice lacking nociceptin receptors. Nature, 394, 577-81.

MARQUEZ, P., BORSE, J., NGUYEN, A.T., HAMID, A. & LUTFY, K. (2008). The role of the opioid receptor-like (ORL1) receptor in motor stimulatory and rewarding actions of buprenorphine and morphine. Neuroscience., 155, 597-602.

MARTI, M., MELA, F., FANTIN, M., ZUCCHINI, S., BROWN, J.M., WITTA, J., DI BENEDETTO, M., BUZAS, B., REINSCHEID, R.K., SALVADORI, S., GUERRINI, R., ROMUALDI, P., CANDELETTI, S., SIMONATO, M., COX, B.M. & MORARI, M. (2005). Blockade of nociceptin/orphanin FQ transmission attenuates symptoms and neurodegeneration associated with Parkinson's disease. J Neurosci, 25, 9591-601.

MARTI, M., MELA, F., GUERRINI, R., CALO, G., BIANCHI, C. & MORARI, M. (2004a). Blockade of nociceptin/orphanin FQ transmission in rat substantia nigra reverses haloperidol-induced akinesia and normalizes nigral glutamate release. J Neurochem, 91, 1501-4.

MARTI, M., MELA, F., VERONESI, C., GUERRINI, R., SALVADORI, S., FEDERICI, M., MERCURI, N.B., RIZZI, A., FRANCHI, G., BEANI, L., BIANCHI, C. & MORARI, M. (2004b). Blockade of nociceptin/orphanin FQ receptor signaling in rat substantia nigra pars reticulata stimulates nigrostriatal dopaminergic transmission and motor behavior. J Neurosci, 24, 6659-66.

MARTI, M., STOCCHI, S., PAGANINI, F., MELA, F., DE RISI, C., CALO, G., GUERRINI, R., BARNES, T.A., LAMBERT, D.G., BEANI, L., BIANCHI, C. & MORARI, M. (2003). Pharmacological profiles of presynaptic nociceptin/orphanin FQ receptors modulating 5-hydroxytryptamine and noradrenaline release in the rat neocortex. Br J Pharmacol, 138, 91-8.

MARTI, M., TRAPELLA, C. & MORARI, M. (2008). The novel nociceptin/orphanin FQ receptor antagonist Trap-101 alleviates experimental parkinsonism through inhibition of the nigro-thalamic pathway: positive interaction with L-DOPA. J Neurochem, 5, 5.

MARTI, M., TRAPELLA, C., VIARO, R. & MORARI, M. (2007). The nociceptin/orphanin FQ receptor antagonist J-113397 and L-DOPA additively attenuate experimental parkinsonism through overinhibition of the nigrothalamic pathway. J Neurosci., 27, 1297-307.

MARTIN-FARDON, R., CICCOCIOPPO, R., MASSI, M. & WEISS, F. (2000). Nociceptin prevents stress-induced ethanol- but not cocaine-seeking behavior in rats. Neuroreport, 11, 1939-43.

MASON, S.L., HO, M., NICHOLSON, J. & MCKNIGHT, A.T. (2001). In vitro characterization of Ac-RYYRWK-NH(2), Ac-RYYRIK-NH(2) and [Phe1Psi(CH(2)-NH)Gly2] nociceptin(1-13)NH(2) at rat native and recombinant ORL(1) receptors. Neuropeptides, 35, 244-56.

MCDONALD, J., BARNES, T.A., CALO, G., GUERRINI, R., ROWBOTHAM, D.J. & LAMBERT, D.G. (2002). Effects of [(pF)Phe(4)]nociceptin/orphanin FQ-(1-13)NH(2) on GTPgamma(35)S binding and cAMP formation in Chinese hamster ovary cells expressing the human nociceptin/orphanin FQ receptor. Eur J Pharmacol, 443, 7-12.

MCDONALD, J., BARNES, T.A., OKAWA, H., WILLIAMS, J., CALO, G., ROWBOTHAM, D.J. & LAMBERT, D.G. (2003a). Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression: studies using the ecdysone-inducible mammalian expression system. Br J Pharmacol, 140, 61-70.

133

MCDONALD, J., CALO, G., GUERRINI, R. & LAMBERT, D.G. (2003b). UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPgamma(35)S binding studies. Naunyn Schmiedebergs Arch Pharmacol, 367, 183-7.

MCLEOD, R.L., BOLSER, D.C., JIA, Y., PARRA, L.E., MUTTER, J.C., WANG, X., TULSHIAN, D.B., EGAN, R.W. & HEY, J.A. (2002). Antitussive effect of nociceptin/orphanin FQ in experimental cough models. Pulm Pharmacol Ther, 15, 213-6.

MCLEOD, R.L., JIA, Y., FERNANDEZ, X., PARRA, L.E., WANG, X., TULSHIAN, D.B., KISELGOF, E.J., TAN, Z., FAWZI, A.B., SMITH-TORHAN, A., ZHANG, H. & HEY, J.A. (2004). Antitussive profile of the NOP agonist Ro-64-6198 in the guinea pig. Pharmacology., 71, 143-9.

MCLEOD, R.L., PARRA, L.E., MUTTER, J.C., ERICKSON, C.H., CAREY, G.J., TULSHIAN, D.B., FAWZI, A.B., SMITH-TORHAN, A., EGAN, R.W., CUSS, F.M. & HEY, J.A. (2001). Nociceptin inhibits cough in the guinea-pig by activation of ORL(1) receptors. Br J Pharmacol, 132, 1175-8.

MEIS, S. & PAPE, H.C. (1998). Postsynaptic mechanisms underlying responsiveness of amygdaloid neurons to nociceptin/orphanin FQ. J Neurosci, 18, 8133-44.

MENZIES, J.R., GLEN, T., DAVIES, M.R., PATERSON, S.J. & CORBETT, A.D. (1999). In vitro agonist effects of nociceptin and [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) in the mouse and rat colon and the mouse vas deferens. Eur J Pharmacol, 385, 217-23.

MEUNIER, J.C. (1997). Nociceptin/orphanin FQ and the opioid receptor-like ORL1 receptor. Eur J Pharmacol, 340, 1-15.

MEUNIER, J.C., MOLLEREAU, C., TOLL, L., SUAUDEAU, C., MOISAND, C., ALVINERIE, P., BUTOUR, J.L., GUILLEMOT, J.C., FERRARA, P., MONSERRAT, B., MAZARGUIL, H., VASSART, G., PARMENTIER, M. & COSTENTIN, J. (1995). Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature, 377, 532-535.

MILLAN, M.J. (2003). The neurobiology and control of anxious states. Prog Neurobiol., 70, 83-244.

MINAMI, M., HOSOI, Y., TOYA, T., KATAO, Y., MAEKAWA, K., KATSUMATA, S., YABUUCHI, K., ONOGI, T. & SATOH, M. (1993). In situ hybridization study of kappa-opioid receptor mRNA in the rat brain. Neurosci Lett., 162, 161-4.

MINAMI, T., OKUDA-ASHITAKA, E., NISHIUCHI, Y., KIMURA, T., TACHIBANA, S., MORI, H. & ITO, S. (1998). Anti-nociceptive responses produced by human putative counterpart of nocistatin. Br J Pharmacol, 124, 1016-8.

MOGIL, J.S., NESSIM, L.A. & WILSON, S.G. (1999). Strain-dependent effects of supraspinal orphanin FQ/nociceptin on thermal nociceptive sensitivity in mice. Neurosci Lett, 261, 147-50.

MOGIL, J.S. & PASTERNAK, G.W. (2001). The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol Rev, 53, 381-415.

MOLLEREAU, C. & MOULEDOUS, L. (2000). Tissue distribution of the opioid receptor-like (ORL1) receptor. Peptides, 21, 907-17.

MOLLEREAU, C., PARMENTIER, M., MAILLEUX, P., BUTOUR, J.L., MOISAND, C., CHALON, P., CAPUT, D., VASSART, G. & MEUNIER, J.C. (1994). ORL1, a novel member of the opioid receptor family. Cloning, functional expression and localization. FEBS Lett., 341, 33-8.

MOLLEREAU, C., SIMONS, M.J., SOULARUE, P., LINERS, F., VASSART, G., MEUNIER, J.C. & PARMENTIER, M. (1996). Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene. Proc Natl Acad Sci U S A, 93, 8666-70.

MONTEILLET-AGIUS, G., FEIN, J., ANTON, B. & EVANS, C.J. (1998). ORL-1 and mu opioid receptor antisera label different fibers in areas involved in pain processing. J Comp Neurol, 399, 373-83.

134

MONTIEL, J.L., CORNILLE, F., ROQUES, B.P. & NOBLE, F. (1997). Nociceptin/orphanin FQ metabolism: role of aminopeptidase and endopeptidase 24.15. J Neurochem, 68, 354-61.

MORINI, G., DE CARO, G., GUERRINI, R., MASSI, M. & POLIDORI, C. (2005). Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat. Regul Pept, 124, 203-7.

MOULEDOUS, L., TOPHAM, C.M., MAZARGUIL, H. & MEUNIER, J.C. (2000). Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin. J Biol Chem, 275, 29268-74.

MURPHY, N.P. & MAIDMENT, N.T. (1999). Orphanin FQ/nociceptin modulation of mesolimbic dopamine transmission determined by microdialysis. J Neurochem, 73, 179-86.

NAKAGAWA, T., KANEKO, M., INAMURA, S. & SATOH, M. (1999). Intracerebroventricular administration of nocistatin reduces inflammatory hyperalgesia in rats. Neurosci Lett, 265, 64-6.

NAZZARO, C., RIZZI, A., SALVADORI, S., GUERRINI, R., REGOLI, D., ZEILHOFER, H.U. & CALO, G. (2007). UFP-101 antagonizes the spinal antinociceptive effects of nociceptin/orphanin FQ: behavioral and electrophysiological studies in mice. Peptides., 28, 663-9.

NEAL, C.R., JR., MANSOUR, A., REINSCHEID, R., NOTHACKER, H.P., CIVELLI, O., AKIL, H. & WATSON, S.J., JR. (1999a). Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding. J Comp Neurol, 412, 563-605.

NEAL, C.R., JR., MANSOUR, A., REINSCHEID, R., NOTHACKER, H.P., CIVELLI, O. & WATSON, S.J., JR. (1999b). Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol, 406, 503-47.

NEUBIG, R.R., SPEDDING, M., KENAKIN, T. & CHRISTOPOULOS, A. (2003). International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacol Rev, 55, 597-606.

NICHOLSON, J.R., PATERSON, S.J., MENZIES, J.R., CORBETT, A.D. & MCKNIGHT, A.T. (1998). Pharmacological studies on the "orphan" opioid receptor in central and peripheral sites. Can J Physiol Pharmacol., 76, 304-13.

NICOL, B., LAMBERT, D.G., ROWBOTHAM, D.J., OKUDA-ASHITAKA, E., ITO, S., SMART, D. & MCKNIGHT, A.T. (1998). Nocistatin reverses nociceptin inhibition of glutamate release from rat brain slices. Eur J Pharmacol, 356, R1-3.

NISHI, M., HOUTANI, T., NODA, Y., MAMIYA, T., SATO, K., DOI, T., KUNO, J., TAKESHIMA, H., NUKADA, T., NABESHIMA, T., YAMASHITA, T., NODA, T. & SUGIMOTO, T. (1997). Unrestrained nociceptive response and disregulation of hearing ability in mice lacking the nociceptin/orphaninFQ receptor. Embo J, 16, 1858-64.

NISHI, M., TAKESHIMA, H., MORI, M., NAKAGAWARA, K. & TAKEUCHI, T. (1994). Structure and chromosomal mapping of genes for the mouse kappa-opioid receptor and an opioid receptor homologue (MOR-C). Biochem Biophys Res Commun., 205, 1353-7.

NOBLE, F. & ROQUES, B.P. (1997). Association of aminopeptidase N and endopeptidase 24.15 inhibitors potentiate behavioral effects mediated by nociceptin/orphanin FQ in mice. FEBS Lett, 401, 227-9.

NODA, Y., MAMIYA, T., NABESHIMA, T., NISHI, M., HIGASHIOKA, M. & TAKESHIMA, H. (1998). Loss of antinociception induced by naloxone benzoylhydrazone in nociceptin receptor-knockout mice. J Biol Chem, 273, 18047-51.

NOTHACKER, H.P., REINSCHEID, R.K., MANSOUR, A., HENNINGSEN, R.A., ARDATI, A., MONSMA, F.J., JR., WATSON, S.J. & CIVELLI, O. (1996). Primary structure and tissue distribution of the orphanin FQ precursor. Proc Natl Acad Sci U S A, 93, 8677-82.

O'DONNELL, A.M., ELLIS, L.M., RIEDL, M.S., ELDE, R.P. & MAWE, G.M. (2001). Distribution and chemical coding of orphanin FQ/nociceptin-immunoreactive neurons in the myenteric plexus of guinea pig intestines and sphincter of Oddi. J Comp Neurol, 430, 1-11.

135

OBARA, I., PRZEWLOCKI, R. & PRZEWLOCKA, B. (2005). Spinal and local peripheral antiallodynic activity of Ro64-6198 in neuropathic pain in the rat. Pain., 116, 17-25.

OKADA, K., SUJAKU, T., CHUMAN, Y., NAKASHIMA, R., NOSE, T., COSTA, T., YAMADA, Y., YOKOYAMA, M., NAGAHISA, A. & SHIMOHIGASHI, Y. (2000). Highly potent nociceptin analog containing the Arg-Lys triple repeat. Biochem Biophys Res Commun, 278, 493-8.

OKUDA-ASHITAKA, E., MINAMI, T., TACHIBANA, S., YOSHIHARA, Y., NISHIUCHI, Y., KIMURA, T. & ITO, S. (1998). Nocistatin, a peptide that blocks nociceptin action in pain transmission. Nature, 392, 286-9.

OLIANAS, M.C. & ONALI, P. (2002). Pharmacological properties of nociceptin/orphanin FQ-induced stimulation and inhibition of cyclic AMP formation in distinct layers of rat olfactory bulb. Br J Pharmacol, 135, 233-8.

OLSZEWSKI, P.K. & LEVINE, A.S. (2004). Minireview: Characterization of influence of central nociceptin/orphanin FQ on consummatory behavior. Endocrinology, 145, 2627-32.

OLSZEWSKI, P.K., SHAW, T.J., GRACE, M.K., BILLINGTON, C.J. & LEVINE, A.S. (2000). Nocistatin inhibits food intake in rats. Brain Res, 872, 181-7.

OSINSKI, M.A., BASS, P. & GAUMNITZ, E.A. (1999a). Peripheral and central actions of orphanin FQ (nociceptin) on murine colon. Am J Physiol, 276, G125-31.

OSINSKI, M.A., PAMPUSCH, M.S., MURTAUGH, M.P. & BROWN, D.R. (1999b). Cloning, expression and functional role of a nociceptin/orphanin FQ receptor in the porcine gastrointestinal tract. Eur J Pharmacol, 365, 281-9.

OUAGAZZAL, A.M., MOREAU, J.L., PAULY-EVERS, M. & JENCK, F. (2003). Impact of environmental housing conditions on the emotional responses of mice deficient for nociceptin/orphanin FQ peptide precursor gene. Behav Brain Res, 144, 111-7.

OZAKI, S., KAWAMOTO, H., ITO, Y., HAYASHI, K., IWASAWA, Y. & HIRANO, K. (1998). New 2-oxoimidazole derivatives - are nociceptin receptor binding inhibitors useful e.g. as analgesics and for amelioreting brain function. Japan.

OZAKI, S., KAWAMOTO, H., ITOH, Y., MIYAJI, M., AZUMA, T., ICHIKAWA, D., NAMBU, H., IGUCHI, T., IWASAWA, Y. & OHTA, H. (2000). In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist. Eur J Pharmacol, 402, 45-53.

PALOMBI, G. & RONZONI, S. (2003). Preparation of benzosuberonylpiperidine compounds. PAN, Y.X., XU, J., WAN, B.L., ZUCKERMAN, A. & PASTERNAK, G.W. (1998). Identification and

differential regional expression of KOR-3/ORL-1 gene splice variants in mouse brain. FEBS Lett, 435, 65-8.

PATON, W.D.M. (1957). The action of morphine and related substances on contraction and on acetylcoline output of coaxially stimulated guinea pig ileum. Br J Pharmacol, 12, 119-127.

PELUSO, J., LAFORGE, K.S., MATTHES, H.W., KREEK, M.J., KIEFFER, B.L. & GAVERIAUX-RUFF, C. (1998). Distribution of nociceptin/orphanin FQ receptor transcript in human central nervous system and immune cells. J Neuroimmunol, 81, 184-92.

PHENG, L.H., CALO, G., GUERRINI, R. & REGOLI, D. (2000). [Nphe(1)]nociceptin-(1-13)NH(2) selectively antagonizes nociceptin effects in the rabbit isolated ileum. Eur J Pharmacol, 397, 383-8.

POLIDORI, C., CALO, G., CICCOCIOPPO, R., GUERRINI, R., REGOLI, D. & MASSI, M. (2000a). Pharmacological characterization of the nociceptin receptor mediating hyperphagia: identification of a selective antagonist. Psychopharmacology (Berl), 148, 430-7.

POLIDORI, C., DE CARO, G. & MASSI, M. (2000b). The hyperphagic effect of nociceptin/orphanin FQ in rats. Peptides, 21, 1051-62.

POMONIS, J.D., BILLINGTON, C.J. & LEVINE, A.S. (1996). Orphanin FQ, agonist of orphan opioid receptor ORL1, stimulates feeding in rats. Neuroreport, 8, 369-71.

136

PRZYDZIAL, M.J. & HEISLER, L.K. (2008). Nociceptin/orphanin FQ peptide receptor as a therapeutic target for obesity. Mini Rev Med Chem., 8, 796-811.

REDROBE, J.P., CALO, G., GUERRINI, R., REGOLI, D. & QUIRION, R. (2000). [Nphe(1)]-Nociceptin (1-13)-NH(2), a nociceptin receptor antagonist, reverses nociceptin-induced spatial memory impairments in the Morris water maze task in rats. Br J Pharmacol, 131, 1379-84.

REDROBE, J.P., CALO, G., REGOLI, D. & QUIRION, R. (2002). Nociceptin receptor antagonists display antidepressant-like properties in the mouse forced swimming test. Naunyn Schmiedebergs Arch Pharmacol, 365, 164-7.

REINSCHEID, R.K., ARDATI, A., MONSMA, F.J., JR. & CIVELLI, O. (1996). Structure-activity relationship studies on the novel neuropeptide orphanin FQ. J Biol Chem, 271, 14163-8.

REINSCHEID, R.K. & CIVELLI, O. (2002). The orphanin FQ/nociceptin knockout mouse: a behavioral model for stress responses. Neuropeptides., 36, 72-6.

REINSCHEID, R.K., HIGELIN, J., HENNINGSEN, R.A., MONSMA, F.J., JR. & CIVELLI, O. (1998). Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities. J Biol Chem, 273, 1490-5.

REINSCHEID, R.K., NOTHACKER, H.P., BOURSON, A., ARDATI, A., HENNINGSEN, R.A., BUNZOW, J.R., GRANDY, D.K., LANGEN, H., MONSMA, F.J., JR. & CIVELLI, O. (1995). Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science, 270, 792-4.

REYNOLDS, S.M., MACKENZIE, A.J., SPINA, D. & PAGE, C.P. (2004). The pharmacology of cough. Trends Pharmacol Sci., 25, 569-76.

RIEDL, M., SHUSTER, S., VULCHANOVA, L., WANG, J., LOH, H.H. & ELDE, R. (1996). Orphanin FQ/nociceptin-immunoreactive nerve fibers parallel those containing endogenous opioids in rat spinal cord. Neuroreport, 7, 1369-72.

RIZZI, A., BIGONI, R., CALO, G., GUERRINI, R., SALVADORI, S. & REGOLI, D. (1999a). [Nphe(1)]nociceptin-(1-13)-NH(2) antagonizes nociceptin effects in the mouse colon. Eur J Pharmacol, 385, R3-5.

RIZZI, A., BIGONI, R., MARZOLA, G., GUERRINI, R., SALVADORI, S., REGOLI, D. & CALO, G. (2001a). Characterization of the locomotor activity-inhibiting effect of nociceptin/orphanin FQ in mice. Naunyn Schmiedebergs Arch Pharmacol, 363, 161-5.

RIZZI, A., BIGONI, R., MARZOLA, G., GUERRINI, R., SALVADORI, S., REGOLI, D. & CALO, G. (2000). The nociceptin/orphanin FQ receptor antagonist, [Nphe1]NC(1-13)NH2, potentiates morphine analgesia. Neuroreport, 11, 2369-72.

RIZZI, A., CALO, G., TREVISANI, M., TOGNETTO, M., FABBRI, L., MAPP, C., GUERRINI, R., SALVADORI, S., REGOLI, D. & GEPPETTI, P. (1999b). Nociceptin receptor activation inhibits tachykinergic non adrenergic non cholinergic contraction of guinea pig isolated bronchus. Life Sci, 64, PL157-63.

RIZZI, A., GAVIOLI, E.C., MARZOLA, G., SPAGNOLO, B., ZUCCHINI, S., CICCOCIOPPO, R., TRAPELLA, C., REGOLI, D. & CALO, G. (2007a). Pharmacological Characterization of the Nociceptin/Orphanin FQ Receptor Antagonist SB-612111 [(-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9 -tetrahydro-5H-benzocyclohepten-5-ol]: In Vivo Studies. J Pharmacol Exp Ther., 321, 968-74.

RIZZI, A., MARZOLA, G., BIGONI, R., GUERRINI, R., SALVADORI, S., MOGIL, J.S., REGOLI, D. & CALO, G. (2001b). Endogenous nociceptin signaling and stress-induced analgesia. Neuroreport, 12, 3009-13.

RIZZI, A., MARZOLA, G., GUERRINI, R., SALVADORI, S., REGOLI, D. & CALO, G. (2008). Antinociceptive effects of nociceptin/orphanin FQ receptor agonists in the mouse writhing test In Society of Neuroscience. pp. 269.15/GG30. Washington, DC. 15-19 Nov.

RIZZI, A., NAZZARO, C., MARZOLA, G.G., ZUCCHINI, S., TRAPELLA, C., GUERRINI, R., ZEILHOFER, H.U., REGOLI, D. & CALO, G. (2006). Endogenous nociceptin/orphanin FQ signalling

137

produces opposite spinal antinociceptive and supraspinal pronociceptive effects in the mouse formalin test: pharmacological and genetic evidences. Pain., 124, 100-8.

RIZZI, A., RIZZI, D., MARZOLA, G., REGOLI, D., LARSEN, B.D., PETERSEN, J.S. & CALO, G. (2002a). Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand, ZP120: in vitro and in vivo studies in mice. Br J Pharmacol, 137, 369-74.

RIZZI, A., SALIS, M.B., CICCOCIOPPO, R., MARZOLA, G., BIGONI, R., GUERRINI, R., MASSI, M., MADEDDU, P., SALVADORI, S., REGOLI, D. & CALO, G. (2002b). Pharmacological characterisation of [(pX)Phe4]nociceptin(1-13)NH2 analogues. 2. In vivo studies. Naunyn Schmiedebergs Arch Pharmacol, 365, 450-6.

RIZZI, A., SPAGNOLO, B., WAINFORD, R.D., FISCHETTI, C., GUERRINI, R., MARZOLA, G., BALDISSEROTTO, A., SALVADORI, S., REGOLI, D., KAPUSTA, D.R. & CALO, G. (2007b). In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor. Peptides., 28, 1240-51.

RIZZI, D., BIGONI, R., RIZZI, A., JENCK, F., WICHMANN, J., GUERRINI, R., REGOLI, D. & CALO, G. (2001c). Effects of Ro 64-6198 in nociceptin/orphanin FQ-sensitive isolated tissues. Naunyn Schmiedebergs Arch Pharmacol, 363, 551-5.

RIZZI, D., RIZZI, A., BIGONI, R., CAMARDA, V., MARZOLA, G., GUERRINI, R., DE RISI, C., REGOLI, D. & CALO, G. (2002c). [Arg(14),Lys(15)]nociceptin, a highly potent agonist of the nociceptin/orphanin FQ receptor: in vitro and in vivo studies. J Pharmacol Exp Ther, 300, 57-63.

ROBERTO, M. & SIGGINS, G.R. (2006). Nociceptin/orphanin FQ presynaptically decreases GABAergic transmission and blocks the ethanol-induced increase of GABA release in central amygdala. Proc Natl Acad Sci U S A., 103, 9715-20.

ROMINGER, A., FORSTER, S., ZENTNER, J., DOOLEY, D.J., MCKNIGHT, A.T., FEUERSTEIN, T.J., JACKISCH, R. & VLASKOVSKA, M. (2002). Comparison of the ORL1 receptor-mediated inhibition of noradrenaline release in human and rat neocortical slices. Br J Pharmacol, 135, 800-6.

ROOZENDAAL, B., LENGVILAS, R., MCGAUGH, J.L., CIVELLI, O. & REINSCHEID, R.K. (2007). Orphanin FQ/nociceptin interacts with the basolateral amygdala noradrenergic system in memory consolidation. Learn Mem., 14, 29-35. Print 2007 Jan-Feb.

ROSSI, G.C., MATHIS, J.P. & PASTERNAK, G.W. (1998). Analgesic activity of orphanin FQ2, murine prepro-orphanin FQ141-157 in mice. Neuroreport, 9, 1165-8.

RUIZ-VELASCO, V., TRAPELLA, C., CALO, G. & MARGAS, W. (2008). Pharmacology of constitutively active NOP opioid receptors heterologously expressed in rat sympathetic neurons. In Society for Neuroscience. Washington, DC, USA.

SAKOORI, K. & MURPHY, N.P. (2004). Central administration of nociceptin/orphanin FQ blocks the acquisition of conditioned place preference to morphine and cocaine, but not conditioned place aversion to naloxone in mice. Psychopharmacology (Berl), 172, 129-36.

SAKURADA, C., SAKURADA, S., ORITO, T., TAN-NO, K. & SAKURADA, T. (2002). Degradation of nociceptin (orphanin FQ) by mouse spinal cord synaptic membranes is triggered by endopeptidase-24.11: an in vitro and in vivo study. Biochem Pharmacol, 64, 1293-303.

SANDIN, J., GEORGIEVA, J., SCHOTT, P.A., OGREN, S.O. & TERENIUS, L. (1997). Nociceptin/orphanin FQ microinjected into hippocampus impairs spatial learning in rats. Eur J Neurosci, 9, 194-7.

SCHILD, H.O. (1997). pA, a new scale for the measurement of drug antagonism. 1947. Br J Pharmacol., 120, 29-46; discussion 27-8.

SCHLICKER, E. & MORARI, M. (2000). Nociceptin/orphanin FQ and neurotransmitter release in the central nervous system. Peptides, 21, 1023-9.

SCHULZ, R., FAASE, E., WUSTER, M. & HERZ, A. (1979). Selective receptors for beta-endorphin on the rat vas deferens. Life Sci., 24, 843-9.

138

SCHULZ, S., SCHREFF, M., NUSS, D., GRAMSCH, C. & HOLLT, V. (1996). Nociceptin/orphanin FQ and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions. Neuroreport., 7, 3021-5.

SEKI, T., AWAMURA, S., KIMURA, C., IDE, S., SAKANO, K., MINAMI, M., NAGASE, H. & SATOH, M. (1999). Pharmacological properties of TRK-820 on cloned mu-, delta- and kappa-opioid receptors and nociceptin receptor. Eur J Pharmacol., 376, 159-67.

SHINKAI, H., ITO, T., IIDA, T., KITAO, Y., YAMADA, H. & UCHIDA, I. (2000). 4-Aminoquinolines: novel nociceptin antagonists with analgesic activity. J Med Chem, 43, 4667-77.

SHIRASAKA, T., KUNITAKE, T., KATO, K., TAKASAKI, M. & KANNAN, H. (1999). Nociceptin modulates renal sympathetic nerve activity through a central action in conscious rats. Am J Physiol, 277, R1025-32.

SHOBLOCK, J.R. (2007). The pharmacology of Ro 64-6198, a systemically active, nonpeptide NOP receptor (opiate receptor-like 1, ORL-1) agonist with diverse preclinical therapeutic activity. CNS Drug Rev., 13, 107-36.

SIM, L.J. & CHILDERS, S.R. (1997). Anatomical distribution of mu, delta, and kappa opioid- and nociceptin/orphanin FQ-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding in guinea pig brain. J Comp Neurol, 386, 562-72.

SIMONSEN, U., LAURSEN, B.E. & PETERSEN, J.S. (2008). ZP120 causes relaxation by pre-junctional inhibition of noradrenergic neurotransmission in rat mesenteric resistance arteries. Br J Pharmacol., 153, 1185-94.

SPAGNOLO, B., CARRA, G., FANTIN, M., FISCHETTI, C., HEBBES, C., MCDONALD, J., BARNES, T.A., RIZZI, A., TRAPELLA, C., FANTON, G., MORARI, M., LAMBERT, D.G., REGOLI, D. & CALO, G. (2007). Pharmacological Characterization of the Nociceptin/Orphanin FQ Receptor Antagonist SB-612111 [(-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9 -tetrahydro-5H-benzocyclohepten-5-ol]: In Vitro Studies. J Pharmacol Exp Ther., 321, 961-7.

SPAMPINATO, S. & BAIULA, M. (2006). Agonist-regulated endocytosis and desensitization of the human nociceptin receptor. Neuroreport., 17, 173-7.

SUYAMA, H., KAWAMOTO, M., GAUS, S. & YUGE, O. (2003). Effect of JTC-801 (nociceptin antagonist) on neuropathic pain in a rat model. Neurosci Lett, 351, 133-6.

TADA, H., NAKAGAWA, K., YAMAMURA, T. & TAKAHASHI, T. (2002). Antagonistic effects of CompB on orphanin FQ-induced colonic contractions in rats. Eur J Pharmacol, 454, 53-8.

TANIGUCHI, H., YOMOTA, E., NOGI, K., ONODA, Y. & IKEZAWA, K. (1998). The effect of nociceptin, an endogenous ligand for the ORL1 receptor, on rat colonic contraction and transit. Eur J Pharmacol, 353, 265-71.

TATEMOTO, K. & MUTT, V. (1980). Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature., 285, 417-8.

TERENIUS, L., SANDIN, J. & SAKURADA, T. (2000). Nociceptin/orphanin FQ metabolism and bioactive metabolites. Peptides, 21, 919-22.

THOMSEN, C. & HOHLWEG, R. (2000a). (8-Naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4. 5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) is a novel potent nociceptin receptor agonist. Br J Pharmacol, 131, 903-8.

THOMSEN, C., VALSBORG, J.S., PLATOU, J., MARTIN, J., FOGED, C., JOHANSEN, N.L., OLSEN, U.B. & MADSEN, K. (2000b). [3H]ac-RYYRWK-NH2, a novel specific radioligand for the nociceptin/orphanin FQ receptor. Naunyn Schmiedebergs Arch Pharmacol, 362, 538-45.

TIAN, J.H., XU, W., FANG, Y., MOGIL, J.S., GRISEL, J.E., GRANDY, D.K. & HAN, J.S. (1997a). Bidirectional modulatory effect of orphanin FQ on morphine-induced analgesia: antagonism in brain and potentiation in spinal cord of the rat. Br J Pharmacol, 120, 676-80.

139

TIAN, J.H., XU, W., ZHANG, W., FANG, Y., GRISEL, J.E., MOGIL, J.S., GRANDY, D.K. & HAN, J.S. (1997b). Involvement of endogenous orphanin FQ in electroacupuncture-induced analgesia. Neuroreport, 8, 497-500.

TOPHAM, C.M., MOULEDOUS, L., PODA, G., MAIGRET, B. & MEUNIER, J.C. (1998). Molecular modelling of the ORL1 receptor and its complex with nociceptin. Protein Eng., 11, 1163-79.

TRAPELLA, C., GUERRINI, R., PICCAGLI, L., CALO, G., CARRA, G., SPAGNOLO, B., RUBINI, S., FANTON, G., HEBBES, C., MCDONALD, J., LAMBERT, D.G., REGOLI, D. & SALVADORI, S. (2006). Identification of an achiral analogue of J-113397 as potent nociceptin/orphanin FQ receptor antagonist. Bioorg Med Chem, 14, 692-704.

TROMBELLA, S., VERGURA, R., FALZARANO, S., GUERRINI, R., CALO, G. & SPISANI, S. (2005). Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation. Peptides., 26, 1497-502.

UCHIYAMA, H., YAMAGUCHI, T., TODA, A., HIRANITA, T., WATANABE, S. & EYANAGI, R. (2008). Involvement of the GABA/benzodiazepine receptor in the axiolytic-like effect of nociceptin/orphanin FQ. Eur J Pharmacol., 590, 185-9.

UEDA, H., INOUE, M., TAKESHIMA, H. & IWASAWA, Y. (2000). Enhanced spinal nociceptin receptor expression develops morphine tolerance and dependence. J Neurosci, 20, 7640-7.

VARANI, K., RIZZI, A., CALO, G., BIGONI, R., TOTH, G., GUERRINI, R., GESSI, S., SALVADORI, S., BOREA, P.A. & REGOLI, D. (1999). Pharmacology of [Tyr1]nociceptin analogs: receptor binding and bioassay studies. Naunyn Schmiedebergs Arch Pharmacol, 360, 270-7.

VARTY, G.B., HYDE, L.A., HODGSON, R.A., LU, S.X., MCCOOL, M.F., KAZDOBA, T.M., DEL VECCHIO, R.A., GUTHRIE, D.H., POND, A.J., GRZELAK, M.E., XU, X., KORFMACHER, W.A., TULSHIAN, D., PARKER, E.M. & HIGGINS, G.A. (2005). Characterization of the nociceptin receptor (ORL-1) agonist, Ro64-6198, in tests of anxiety across multiple species. Psychopharmacology (Berl). 182, 132-43.

VARTY, G.B., LU, S.X., MORGAN, C.A., COHEN-WILLIAMS, M.E., HODGSON, R.A., SMITH-TORHAN, A., ZHANG, H., FAWZI, A.B., GRAZIANO, M.P., HO, G.D., MATASI, J., TULSHIAN, D., COFFIN, V.L. & CAREY, G.J. (2008). The anxiolytic-like effects of the novel, orally active nociceptin opioid receptor agonist 8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510). J Pharmacol Exp Ther., 326, 672-82.

VAUGHAN, C.W. & CHRISTIE, M.J. (1996). Increase by the ORL1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurones. Br J Pharmacol, 117, 1609-11.

VAUGHAN, C.W., INGRAM, S.L. & CHRISTIE, M.J. (1997). Actions of the ORL1 receptor ligand nociceptin on membrane properties of rat periaqueductal gray neurons in vitro. J Neurosci, 17, 996-1003.

VERGURA, R., BALBONI, G., SPAGNOLO, B., GAVIOLI, E.C., LAMBERT, D.G., MCDONALD, J., TRAPELLA, C., REGOLI, D., SALVADORI, S. & CALO', G. (2008). Anxiolytic- and antidepressant-like activities of UFP-512, a novel selective delta opioid receptor agonist. Peptides, 29, 93-103.

VIARO, R., SANCHEZ-PERNAUTE, R., MARTI, M., TRAPELLA, C., ISACSON, O. & MORARI, M. (2008). Nociceptin/orphanin FQ receptor blockade attenuates MPTP-induced parkinsonism. Neurobiol Dis., 30, 430-8.

VISANJI, N.P., DE BIE, R.M., JOHNSTON, T.H., MCCREARY, A.C., BROTCHIE, J.M. & FOX, S.H. (2008). The nociceptin/orphanin FQ (NOP) receptor antagonist J-113397 enhances the effects of levodopa in the MPTP-lesioned nonhuman primate model of Parkinson's disease. Mov Disord., 23, 1922-5.

VITALE, G., ARLETTI, R., RUGGIERI, V., CIFANI, C. & MASSI, M. (2006). Anxiolytic-like effects of nociceptin/orphanin FQ in the elevated plus maze and in the conditioned defensive burying test in rats. Peptides., 27, 2193-200.

140

VITALE, G., RUGGIERI, V., FRIGERI, C., FILAFERRO, M., CIFANI, C., ZUCCHINI, S., SIMONATO, M., MASSI, M. (2008). Chronic treatment with [Nphe1, Arg14, Lys15] N/OFQ-NH2 (UFP-101), a potent NOP receptor antagonist, reverses behavioral and biochemical effects in the rat chronic mild stress.In EOC-ENC. pp. 67 Abstract Book Ferrara.

WALKER, J.R., SPINA, M., TERENIUS, L. & KOOB, G.F. (1998). Nociceptin fails to affect heroin self-administration in the rat. Neuroreport, 9, 2243-7.

WANG, J.B., JOHNSON, P.S., IMAI, Y., PERSICO, A.M., OZENBERGER, B.A., EPPLER, C.M. & UHL, G.R. (1994). cDNA cloning of an orphan opiate receptor gene family member and its splice variant. FEBS Lett., 348, 75-9.

WANG, J.L., ZHU, C.B., CAO, X.D. & WU, G.C. (1999). Distinct effect of intracerebroventricular and intrathecal injections of nociceptin/orphanin FQ in the rat formalin test. Regul Pept, 79, 159-63.

WATSON, B.T., HOHLWEG, R. & THOMSEN, C. (1999). Novel 1,3,8-triazaspiro[4,5]deconones with affinity for opiod receptor subtypes.

WICHMANN, J., ADAM, G., ROVER, S., CESURA, A.M., DAUTZENBERG, F.M. & JENCK, F. (1999). 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists. Bioorg Med Chem Lett, 9, 2343-8.

WICK, M.J., MINNERATH, S.R., LIN, X., ELDE, R., LAW, P.Y. & LOH, H.H. (1994). Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors. Brain Res Mol Brain Res., 27, 37-44.

WICK, M.J., MINNERATH, S.R., ROY, S., RAMAKRISHNAN, S. & LOH, H.H. (1995). Expression of alternate forms of brain opioid 'orphan' receptor mRNA in activated human peripheral blood lymphocytes and lymphocytic cell lines. Brain Res Mol Brain Res., 32, 342-7.

WILLIAMS, J.P., THOMPSON, J.P., YOUNG, S.P., GOLD, S.J., MCDONALD, J., ROWBOTHAM, D.J. & LAMBERT, D.G. (2008). Nociceptin and urotensin-II concentrations in critically ill patients with sepsis. Br J Anaesth., 100, 810-4.

WISE, A., JUPE, S.C. & REES, S. (2004). The identification of ligands at orphan G-protein coupled receptors. Annu Rev Pharmacol Toxicol, 44, 43-66.

WISE, R.A. (1989). Opiate reward: sites and substrates. Neurosci Biobehav Rev., 13, 129-33. WITTA, J., PALKOVITS, M., ROSENBERGER, J. & COX, B.M. (2004). Distribution of

nociceptin/orphanin FQ in adult human brain. Brain Res, 997, 24-9. WNENDT, S., KRUGER, T., JANOCHA, E., HILDEBRANDT, D. & ENGLBERGER, W. (1999). Agonistic

effect of buprenorphine in a nociceptin/OFQ receptor-triggered reporter gene assay. Mol Pharmacol, 56, 334-8.

WU, Y., FANG, L.B. & YANG, Q.H. (2000). Nociceptin inhibits electric field stimulation-induced cholinergic constrictions in rat airways. Acta Pharmacol Sin, 21, 945-8.

XU, I.S., GRASS, S., CALO, G., GUERRINI, R., WIESENFELD-HALLIN, Z. & XU, X.J. (2002). Intrathecal [Nphe1]nociceptin( 1-13)NH2 selectively reduces the spinal inhibitory effect of nociceptin. Life Sci, 70, 1151-7.

XU, I.S., WIESENFELD-HALLIN, Z. & XU, X.J. (1998). [Phe1psi(CH2-NH)Gly2]-nociceptin-(1-13)NH2, a proposed antagonist of the nociceptin receptor, is a potent and stable agonist in the rat spinal cord. Neurosci Lett, 249, 127-30.

XU, X.J., HAO, J.X. & WIESENFELD-HALLIN, Z. (1996). Nociceptin or antinociceptin: potent spinal antinociceptive effect of orphanin FQ/nociceptin in the rat. Neuroreport, 7, 2092-4.

YAMADA, H., NAKAMOTO, H., SUZUKI, Y., ITO, T. & AISAKA, K. (2002). Pharmacological profiles of a novel opioid receptor-like1 (ORL(1)) receptor antagonist, JTC-801. Br J Pharmacol, 135, 323-32.

YAMAMOTO, T., SAKASHITA, Y. & NOZAKI-TAGUCHI, N. (2001). Antagonism of ORLI receptor produces an algesic effect in the rat formalin test. Neuroreport, 12, 1323-7.

141

YAN-YU, L. & CHIOU, L.C. (2008). Effect of compound 24, a novel nociceptin/orphanin FQ (NOP) receptor antagonist, on NOP receptor-mediated k+ channel activation in rat periaqueductal gray slices. In Society for Neuroscience. Washington, DC, USA.

YASUDA, K., RAYNOR, K., KONG, H., BREDER, C.D., TAKEDA, J., REISINE, T. & BELL, G.I. (1993). Cloning and functional comparison of kappa and delta opioid receptors from mouse brain. Proc Natl Acad Sci U S A, 90, 6736-6740.

YAZDANI, A., TAKAHASHI, T., BAGNOL, D., WATSON, S.J. & OWYANG, C. (1999). Functional significance of a newly discovered neuropeptide, orphanin FQ, in rat gastrointestinal motility. Gastroenterology, 116, 108-17.

YU, T.P., FEIN, J., PHAN, T., EVANS, C.J. & XIE, C.W. (1997). Orphanin FQ inhibits synaptic transmission and long-term potentiation in rat hippocampus. Hippocampus, 7, 88-94.

ZARATIN, P.F., PETRONE, G., SBACCHI, M., GARNIER, M., FOSSATI, C., PETRILLO, P., RONZONI, S., GIARDINA, G.A. & SCHEIDELER, M.A. (2004). Modification of nociception and morphine tolerance by the selective opiate receptor-like orphan receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9- tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). J Pharmacol Exp Ther, 308, 454-61.

ZAVERI, N. (2003). Peptide and nonpeptide ligands for the nociceptin/orphanin FQ receptor ORL1: research tools and potential therapeutic agents. Life Sci, 73, 663-78.

ZAVERI, N., JIANG, F., OLSEN, C., POLGAR, W. & TOLL, L. (2005). Small-molecule agonists and antagonists of the opioid receptor-like receptor (ORL1, NOP): ligand-based analysis of structural factors influencing intrinsic activity at NOP. Aaps J, 7, E345-52.

ZAVERI, N., POLGAR, W.E., OLSEN, C.M., KELSON, A.B., GRUNDT, P., LEWIS, J.W. & TOLL, L. (2001). Characterization of opiates, neuroleptics, and synthetic analogs at ORL1 and opioid receptors. Eur J Pharmacol, 428, 29-36.

ZAVERI, N.T., GREEN, C.J. & TOLL, L. (2000). Transcriptional regulation of the human prepronociceptin gene. Biochem Biophys Res Commun., 276, 710-7.

ZEILHOFER, H.U. & CALO, G. (2003). Nociceptin/orphanin FQ and its receptor--potential targets for pain therapy? J Pharmacol Exp Ther, 306, 423-9.

ZHANG, C., MILLER, W., VALENZANO, K.J. & KYLE, D.J. (2002). Novel, potent ORL-1 receptor agonist peptides containing alpha-Helix-promoting conformational constraints. J Med Chem., 45, 5280-6.

ZHANG, G., MURRAY, T.F. & GRANDY, D.K. (1997). Orphanin FQ has an inhibitory effect on the guinea pig ileum and the mouse vas deferens. Brain Res, 772, 102-6.

ZHU, C.B., CAO, X.D., XU, S.F. & WU, G.C. (1997). Orphanin FQ potentiates formalin-induced pain behavior and antagonizes morphine analgesia in rats. Neurosci Lett, 235, 37-40.

142

List of Publications Full papers

• Spagnolo B., Carrà G., Fantin M., Fischetti C., Hebbes C., McDonald J., Barnes T.A., Rizzi A., Trapella C., Fanton G., Morari M., Lambert D.G., Regoli D., Calò G. Pharmacological characterization of the nociceptin/orphanin FQ receptor antagonist SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol]: in vitro studies. J Pharmacol Exp Ther. 2007;321(3):961-7.

• Rizzi A., Spagnolo B., Wainford R.D., Fischetti C., Guerrini R., Marzola G., Baldisserotto A., Salvadori S., Regoli D., Kapusta D.R., Calo G. In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor. Peptides. 2007;28(6):1240-51.

• Arduin M., Calo’ G., Guerrini R., Spagnolo B., Fischetti C., Trappella C., Marzola E., Regoli D., and Salvadori S. Synthesis and biological activity of Nociceptin / Orphanin FQ analogues substituited in position 7 or 11 with Cα,α-dialkylated amino acids. Bioorg Med Chem. 2007;15(13):4434-43.

• Fantin M., Fischetti C., Trapella C., Morari M. Nocistatin inhibits 5-hydroxytryptamine release in the mouse neocortex via presynaptic Gi/o protein linked pathways. Br J Pharmacol. 2007;152(4):549-55.

• Fischetti C., Rizzi A., Gavioli E.C., Marzola G., Trapella C., Guerrini R., Petersen J.S., Calo G. Further studies on the pharmacological features of the nociceptin/orphanin FQ receptor ligand ZP120. Peptides. 2009; 30:248-55.

• Camarda V., Fischetti C., Anzellotti N., Molinari P., Ambrosio C., Kostenis E., Regoli D., Trapella C., Guerrini R., Severo S., Calo’ G. Pharmacological profile of NOP receptors coupled with calcium signalling via the chimeric protein Gαqi5. Naunyn Schmiedebergs Arch Pharmacol. 2009. in press

• Fischetti C., Camarda V., Rizzi A., Pelà M., Trapella C., R Guerrini R., McDonald J., Lambert D.G., Salvatori S., Regoli D., Calo’ G. Pharmacological characterization of the nociceptin/orphanin FQ receptor non peptide antagonist Compound 24. Submitted to Eur J Pharmacol.

• Trapella C., Fischetti C., Pela’M., Lazzari I., Guerrini R., Calo’ G., Lambert D.G., McDonald J., Regoli D., Salvadori S. Blending of chemical moieties of NOP receptor ligands: identification of a novel antagonist. Submitted to J Med Chem.

Abstracts

• Fischetti C., Fantin M., Morari M. Nocistatin inhibits 5-hydroxytryptamine release in the mouse neocortex via G protein coupled presynaptic receptors. National Congress of the Italian-Swedish for Neuroscience, Ischia (Italy) 1-4 October 2005 (poster presentation).

• Marti M., Mela F., Fischetti C., Morari M. Nociceptin / Orphanin FQ receptor antagonists as a novel therapeutic approach to Parkinson’s disease. National Congress of the Italian-Swedish for Neuroscience, Ischia (Italy) 1-4 October 2005 (poster presentation).

• Fantin M., Fischetti C., Ballini C., Della Corte L., Marti M., Morari M. Striatal NMDA receptors regulate the striato-pallidal pathway. National Congress of the Italian-Swedish for Neuroscience, Ischia (Italy) 1-4 October 2005 (poster presentation).

• Fischetti C., Spagnolo B., Tourwe D., Kaul R., Lubell W.D., Regoli D., Simonin F. and Calo’ G. Effect of the Nociceptin / Orphanin FQ receptor ligand Ac-Cit-D-Cha-Qaa-D-Arg-D-p-Clphe-NH2 in electrically stimulated isolated tissues. European Opioid Conference, Salamanca (Spain), 19-21 April 2006 (poster presentation).

143

• Spagnolo B., Fischetti C., Arduin M., Guerrini R., Trappella C., Regoli D., Salvadori S. and Calo’ G. Nociceptin/Orphanin FQ analogues substituted with alpha helix inducers aminoacids: identification of a novel series of higly potent and selective ligands for the NOP receptor. European Opioid Conference, Salamanca (Spain), 19-21 April 2006 (poster presentation).

• Fischetti C., Spagnolo B., Carrà G., Fantin M., Hebbes C., McDonald J., Barnes T.A., Rizzi A., Marzola G., Trapella C., Fanton G., Morari M., Lambert D.G., Regoli D., Calò G. Pharmacological characterization of the nociceptin/orphanin FQ receptor antagonist SB-612111: in vitro studies. Società italiana di Farmacologia, Cagliari (Italy) 6-9 June 2007 (poster presentation).

• Fischetti C., Rodi D., Pelà M., Camarda V., Rizzi A., Trapella C., McDonald J., Regoli D., Guerrini R., Lambert D.G., Salvadori S., Calo’ G. In vitro and in vivo characterization of the NOP receptor non peptide antagonist Compound 24. Society for Neuroscience, Washington DC, 15-19 November 2008 (poster presentation).

Date post:	12-Oct-2018
Category:	Documents
Upload:	lamthien
View:	212 times
Download:	0 times

CICLO XXI COORDINATORE Prof. Pier Andrea BOREA Fischetti PhD... · 1 List of abbreviations 3...

Documents